Amelioration of heterophile antibody immunosensor interference

ABSTRACT

The invention is directed to methods and devices for reducing interference from heterophile antibodies in an analyte immunoassay. In one embodiment, the invention is to a method comprising the steps of (a) amending a biological sample such as a whole blood sample with non-human IgM or fragments thereof by dissolving into said sample a dry reagent to yield a non-human IgM concentration of at least about 20 μg/mL or equivalent fragment concentration; and (b) performing an electrochemical immunoassay on the amended sample to determine the concentration of said analyte in said sample. Preferably, the sample is amended with IgG or fragments thereof in addition to the IgM of fragments thereof.

FIELD OF THE INVENTION

The present invention relates to reducing or eliminating heterophileantibody immunosensor interference in devices and methods fordetermining the presence or concentration of analytes in liquid samples.In particular, the invention relates to reducing or eliminatingheterophile antibody immunosensor interference by amending biologicalsamples with gamma globulin proteins such as IgM and fragments thereof.

BACKGROUND OF THE INVENTION

A multitude of laboratory immunoassay tests for analytes of interest areperformed on biological samples for diagnosis, screening, diseasestaging, forensic analysis, pregnancy testing, drug testing, and otherreasons. While a few qualitative tests, such as pregnancy tests, havebeen reduced to simple kits for the patient's home use, the majority ofquantitative tests still require the expertise of trained technicians ina laboratory setting using sophisticated instruments. Laboratory testingincreases the cost of analysis and delays the results. In manycircumstances, delay can be detrimental to a patient's condition orprognosis, such as for example the analysis of markers indicatingmyocardial infarction and heart failure. In these and similar criticalsituations, it is advantageous to perform such analyses at thepoint-of-care, accurately, inexpensively, and with a minimum of delay.

Two-site immunoassays, also called sandwich-type immunoassays, are oftenemployed for determining analyte concentration in biological testsamples, and are used, for example, in the point-of-care analytedetection system developed by Abbott Point-of-care Inc. as the i-Stat®system. In a typical two-site enzyme-linked immunosorbent assay (ELISA),one antibody is bound to a solid support to form an “immobilizedantibody” and a second antibody is conjugated or bound to asignal-generating reagent such as an enzyme to form a “signal antibody.”Upon reaction with a sample containing the analyte to be measured, theanalyte becomes “sandwiched” between the immobilized antibody and thesignal antibody. After washing away the sample and any non-specificallybound reagents, the amount of signal antibody remaining on the solidsupport is measured and should be proportional to the amount of analytein the sample.

Many types of immunoassay devices and processes have been described. Onedisposable sensing device for successfully measuring analytes in asample of blood is disclosed by Lauks in U.S. Pat. No. 5,096,669. Otherdevices are disclosed by Davis et al. in U.S. Pat. Nos. 5,628,961 and5,447,440 for a clotting time. These devices employ a reading apparatusand a cartridge that fits into the reading apparatus for the purpose ofmeasuring analyte concentrations and viscosity changes in a sample ofblood as a function of time. U.S. Pat. Nos. 5,096,669; 5,628,961 and5,447,440 are hereby incorporated herein by reference in theirentireties.

Electrochemical detection, in which binding of an analyte directly orindirectly causes a change in the activity of an electroactive speciesadjacent to an electrode, has also been applied to immunoassay. For areview of electrochemical immunoassay, see Laurell et al., Methods inEnzymology, vol. 73, “Electroimmunoassay”, Academic Press, New York,339, 340, 346-348 (1981).

Microfabrication techniques (e.g. photolithography and plasmadeposition) are attractive for construction of multilayered sensorstructures in confined spaces. Methods for microfabrication ofelectrochemical immunosensors, for example on silicon substrates, aredisclosed in U.S. Pat. No. 5,200,051 to Cozzette et al., which is herebyincorporated by reference in its entirety. These include dispensingmethods, methods for attaching biological reagent, e.g. antibodies, tosurfaces including photoformed layers and microparticle latexes, andmethods for performing electrochemical assays.

In an electrochemical immunosensor, the binding of an analyte to itscognate antibody produces a change in the activity of an electroactivespecies at an electrode that is poised at a suitable electrochemicalpotential to cause oxidation or reduction of the electroactive species.There are many arrangements for meeting these conditions. For example,electroactive species may be attached directly to an analyte, or theantibody may be covalently attached to an enzyme that either produces anelectroactive species from an electroinactive substrate, or destroys anelectroactive substrate. See, M. J. Green (1987) Philos. Trans. R. Soc.Lond. B. Biol. Sci. 316:135-142, for a review of electrochemicalimmunosensors.

The concept of differential amperometric measurement is well known inthe electrochemical art, see for example jointly owned Cozzette, U.S.Pat. No. 5,112,455. In addition, a version of a differentialamperometric sensor combination is disclosed in jointly owned Cozzette,U.S. Pat. No. 5,063,081. This patent also discloses the use ofpermselective layers for electrochemical sensors and the use offilm-forming latexes for immobilization of bioactive molecules,incorporated here by reference. The use of poly(vinyl alcohol) (PVA) insensor manufacture is described in U.S. Pat. No. 6,030,827 incorporatedhere by reference. Vikholm (U.S. 2003/0059954A1) teaches antibodiesdirectly attached to a surface with a biomolecule repellant coating,e.g. PVA, the surface in the gaps between antibodies, and Johansson(U.S. Pat. No. 5,656,504) teaches a solid phase, e.g. PVA, withantibodies immobilized thereon. U.S. Pat. Nos. 6,030,827 and 6,379,883teach methods for patterning poly(vinylalcohol) layers and areincorporated by reference in their entirety.

US 20060160164 describes an immunoassay device with an immuno-referenceelectrode, US 20050054078 describes an immunoassay device with improvedsample closure, US 20040018577 describes a multiple hybrid immunoassay,and US 20030170881 (issued as U.S. Pat. No. 7,419,821) describes anapparatus and methods for analyte measurement and immunoassay, all ofwhich are jointly owned and are incorporated here by reference.

With regard to amperometric measurements, there are several means knownin the art for reducing the importance of the non-Faradaic component ofthe signal, thus increasing sensitivity. These include newerelectrochemical methods, e.g. using square wave voltammetry in place ofchronoamperometry, and chemical means, e.g. an alkyl thiol reagent topassivate an electrode surface.

One limitation of conventional assay configurations, however, is thesusceptibility to interference caused by heterophile antibodies that maybe present in the test sample. See, e.g., L. Kricka, “Human Anti-AnimalAntibody Interferences in Immunological Assays,” Clinical Chemistry 45:7at 942-956 (1999). Antibodies employed in commercial immunoassays are inmany cases prepared or “raised” in animals or media of animal origin.Furthermore, many individuals harbor naturally occurring, non-specificantibodies to animal proteins, “endogenous antibodies,” that may bind tothe animal antibody reagents employed in the immunoassay, leading toerroneous results. For example, endogenous antibodies capable of bindingto one or more of the assay reagents pose the potential to generateerroneous test results by cross-linking the reagents, leading tofalse-positive results, or sequestering the reagents, leading tofalse-negative results.

It has been found, for example, that cancer therapy with radiolabelledmurine monoclonal antibodies can lead to the production of humananti-mouse antibodies (HAMA) in the patient. It was subsequently shownthat the presence of HAMA in serum samples taken from those patients,can cause cross-linking of the reagent murine monoclonal antibodies usedin sandwich-type enzyme immunoassays for cancer markers (Boscato et al.,Heterophile antibodies: A problem for all immunoassays, Clin Chem 34,27-33, 1988). In addition, Nicholson et al., (Immunoglobulin inhibitingreagent (IIR): Evaluation of a new method for eliminating spuriouselevation in CA125 caused by HAMA, Intl J Biol Markers 11, 46-49, 1996)demonstrated beneficial results using IIR (Bioreclamation Inc, NY) toeliminate HAMA interference in a CA125 assay. The IIR material isreported to comprise a partially purified preparation of immunoglobulins(IgG, IgM) from several species, principally murine IgG (subtypes IgG2a,IgG2b and IgG3) from Balb/c mice.

U.S. Pat. No. 6,106,779 teaches that nonspecific binding of certainassay reagents to each other and to device components is often a problemin diagnostic assays. This is particularly a problem when an antibodyrecognizes a region of a molecule that is not its antigen. This can thenlead to high background reactions and false positive (or negative) assayresults. Non-specific binding inhibitors that may be used for thisproblem include bovine IgG.

US 20080261242 discusses endogenous human heterophile antibodies andhuman anti-animal antibodies, which have the ability to bind toimmunoglobulins of other species, and are present in the serum or plasmaof more than 10% of patients. These circulating heterophile antibodiesmay interfere with immunoassay measurements. In sandwich immunoassays,these heterophile antibodies can either bridge the capture and detection(diagnostic) antibodies, thereby producing a false-positive signal, orthey may block the binding of the diagnostic antibodies, therebyproducing a false-negative signal. Additionally, in competitiveimmunoassays, the heterophile antibodies may bind to the analyticantibody and inhibit its binding to the analyte. They also may eitherblock or augment the separation of the antibody-analyte complex fromfree analyte, especially when anti-species antibodies are used inseparation systems. As a result, the impact of these heterophileantibody interferences are often difficult to predict.

Several additional methods for removing heterophile antibodies fromsamples are also known and include: (i) heating the specimen in a sodiumacetate buffer, pH 5.0, for 15 minutes at 90 degrees C. followed bycentrifuging at 1200 g for 10 minutes, (ii) precipitation usingpolyethylene glycol (PEG), and (iii) immunoextraction with protein A orprotein G. Clinical guidelines for dealing with the heterophile antibodyissue are also provided by the Clinical and Laboratory StandardsInstitute (CLSI) Immunoassay Interference by Endogenous Antibodies;Proposed Guideline. CLSI document I/LA30-P (ISBN 1-56238-633-6).

Generally, immunoassay manufacturers strive to reduce heterophileinterference by (a) removal or inactivation of the interferingimmunoglobulins from samples, (b) modification of assay antibodies tomake them less prone to react with heterophile antibodies, and (c) useof blocking agents (mostly IgGs) that reduce interference.

However, the need remains for improved processes for amelioratingheterophile antibodies in at least the following areas: (i) immunosensorinterference, most notably in the context of point-of-care testing, (ii)electrochemical immunoassays, (iii) the use of an immunosensor inconjunction with an immuno-reference sensor, (iv) whole bloodimmunoassays, (v) single-use cartridge based immunoassays, (vi)non-sequential immunoassays with only a single wash step, and (vii) dryreagent coatings.

SUMMARY OF THE INVENTION

The present invention relates to the determination of analytes inbiological samples such as blood using electrochemical immunosensors orother ligand/ligand receptor-based biosensors. Specifically, it relatesto improved ways of reducing interference from heterophile antibodies invarious assays, including, for example, cardiovascular markerimmunoassays. The approach involves collecting a sample, e.g., a bloodsample, and then amending the sample, for example, by dissolving a dryreagent comprising either a selected non-human IgM (immunoglobulin M) orfragments thereof, or a defined mixture of non-human IgG and non-humanIgM, or fragments thereof.

In the invention, a sufficient amount of immunoglobulin is used tosubstantially sequester any heterophile antibodies that are present inthe sample. The amount of reagent is generally selected to ensure thatit is sufficient to bind heterophile antibodies at concentrations inwhich they occur in the majority of the population. Alternatively, theamount of reagent can be selected to ensure that heterophile antibodiesabove a predetermined threshold concentration value are substantiallyremoved, i.e., bound to the added immunoglobulin and therefore preventedfrom interfering with the assay. After a period to allow for thisbinding step to occur, it is then possible to perform the immunoassay,e.g., an electrochemical immunoassay, on the amended sample. Theinvention further relates to the use of these sequestering reagents inconjunction with both an immuno-reference sensor and an immunosensor.The present invention is particularly useful for point-of-care bloodtesting, also referred to as bedside testing and near-patient testing.

In a first embodiment, the invention is to a method of reducinginterference from heterophile antibodies in an analyte immunoassay,comprising: amending a whole blood sample with non-human IgM orfragments thereof by dissolving into the sample a dry reagent to yield anon-human IgM concentration of at least 20 μg/mL or equivalent fragmentconcentration; and performing an electrochemical immunoassay on theamended sample to determine the concentration of the analyte in thesample. In a preferred aspect, the method also comprises amending thewhole blood sample with IgG or fragments thereof. The non-human IgG andIgM preferably are murine, caprine or a combination thereof.

The analyte may vary widely but preferably is selected from the group,TnI, TnT, CKMB, myoglobin, BNP, NT-proBNP, and proBNP. In a preferredembodiment, the sample is amended for a predetermined period rangingfrom about 1 minute to about 30 minutes. The dry reagent preferablyfurther comprises a component selected from the group consisting ofbuffer, salt, surfactant, stabilizing agent, a simple carbohydrate, acomplex carbohydrate and combinations thereof. The dry reagent mayfurther comprise an enzyme-labeled antibody (signal antibody) to theanalyte. In another aspect, the method further comprises the step ofamending the amended sample with an enzyme-labeled antibody (signalantibody) to the analyte by dissolving into the amended sample a seconddry reagent comprising the enzyme-labeled antibody, wherein the seconddry reagent is separate from the dry reagent that contains the IgM orfragments thereof. For example, the dry reagent may further comprise thenon-human IgG.

The electrochemical immunoassay preferably is an enzyme-linked sandwichimmunoassay, and preferably is performed by an immunosensor. Thus, theelectrochemical assay preferably is performed with an immobilizedantibody to the analyte on an electrode. The amended sample preferablyfurther comprises an enzyme-labeled antibody to the analyte and iscontacted with an immobilized antibody to the analyte to form a sandwichof the analyte between the immobilized and labeled antibodies, themethod further comprising the steps of washing the sample to a wastechamber and exposing the sandwich to a substrate capable of reactingwith the enzyme to form a product capable of electrochemical detection.In one embodiment, the electrochemical immunoassay is performed by animmunosensor and an immuno-reference sensor. In another embodiment, theelectrochemical immunoassay is an enzyme-linked immunosorbent assay. Themethod is particularly well suited for being performed at the point ofpatient care. For example, the immunoassay may be performed in acartridge comprising an immunosensor, a conduit, a sample entry port anda sample holding chamber. In this aspect, at least a portion of at leastone of the sample entry port, the sample holding chamber, the conduitand the immunosensor may be coated with the dry reagent.

In another embodiment, the invention is directed to a method of reducinginterference from heterophile antibodies in a cardiac troponin Iimmunoassay, comprising: amending a whole blood sample with a mixturecomprising: (i) non-human IgG or IgG fragments, and (ii) non-human IgMor IgM fragments, sufficient to substantially sequester any heterophileantibodies in the sample, wherein the non-human IgM concentration in theamended sample is at least about 20 μg/mL or equivalent IgM fragmentconcentration; and performing an electrochemical immunoassay on theamended sample.

In another embodiment, the invention is directed to a method of reducinginterference from heterophile antibodies in a brain natriuretic peptideimmunoassay, comprising: amending a sample with a mixture comprising:(i) non-human IgG or IgG fragments, and (ii) non-human IgM or IgMfragments, sufficient to substantially sequester any heterophileantibodies in the sample, wherein the non-human IgM concentration in theamended sample is at least about 20 μg/mL or equivalent IgM fragmentconcentration; and performing an electrochemical immunoassay on theamended sample.

In another embodiment, the invention is to a device, e.g., a single usecartridge, for performing an immunoassay of an analyte in a blood samplewith reduced interference from heterophile antibodies, comprising ahousing, an electrochemical immunosensor, a conduit and a sample entryport, wherein the conduit permits a blood sample to pass from the entryport to the immunosensor, and wherein at least one of the housing, theentry port, and the conduit includes a dry reagent coating comprisingnon-human IgM or fragments thereof and optionally IgG or fragmentsthereof, the dry reagent being capable of dissolving into the bloodsample to yield an IgM concentration of at least about 20 μg/mL orequivalent fragment concentration and substantially sequestering anyheterophile antibodies in the sample. In this aspect, the devicepreferably further comprises a metering system for metering an initialblood sample to form a metered blood sample. The device may alsocomprise an immuno-reference sensor. The immunosensor preferablycomprises an immobilized antibody to the analyte on an electrode.

In one aspect, the dry reagent further comprises a component selectedfrom the group consisting of buffer, salt, surfactant, stabilizingagent, a simple carbohydrate, a complex carbohydrate and combinationsthereof. Optionally, the dry reagent further comprises an enzyme-labeledantibody to the analyte. In an alternative embodiment, the devicefurther comprises a second dry reagent comprising an enzyme-labeledantibody to the analyte, wherein the second dry reagent is separate fromthe dry reagent that comprises the IgM or fragments thereof. The devicepreferably further comprises a wash fluid capable of washing the sampleto a waste chamber. The wash fluid may comprise a substrate capable ofreacting at the immunosensor to form a product capable ofelectrochemical detection.

In another embodiment, the invention is directed to a method of reducinginterference from heterophile antibodies in an analyte immunoassaycomprising: amending a biological sample with IgM or fragments thereofand optionally IgG or fragments thereof to yield a non-human IgMconcentration of at least about 20 μg/mL or equivalent fragmentconcentration; and performing an immunoassay on the amended sample todetermine the concentration of the analyte in the sample. For example,the biological sample may be amended by dissolving into the sample a dryreagent comprising one or more of IgM, IgM fragments, IgG or IgGfragments. The method preferably further comprises the step of amendingthe biological sample with IgG or fragments thereof. The biologicalsample, for example, may be selected from the group consisting of wholeblood, serum, plasma, urine and diluted forms thereof. The immunoassaymethod preferably is selected from the group consisting ofelectrochemical, amperometric, potentiometric, absorbance, fluorescenceand luminescence.

In another embodiment, the invention is to a method of reducingheterophile antibody interference in an analyte immunoassay device,comprising: adding IgM or fragments thereof and IgG or fragments thereofto a biological sample in an amount sufficient to substantiallysequester any heterophile antibodies in the sample and forming anamended sample, wherein the IgM or fragments thereof and the IgG orfragments thereof are added at a weight ratio greater than 0.004, e.g.,greater than 0.02, greater than 0.05 or greater than 0.1; and performingan electrochemical immunoassay on the amended sample to determine theconcentration of the analyte in the amended sample. After the addingstep, the IgM, for example, may be present in the sample in aconcentration of at least 20 μg/mL. Alternatively, the IgM fragments arepresent in the sample in a concentration equivalent to an IgMconcentration of at least about 20 μg/mL. In one aspect, the addingcomprises dissolving the IgM or the fragments thereof into the samplefrom a dry reagent coating contained in the immunoassay device.Alternatively, the adding may comprise dissolving the IgM or thefragments thereof into the sample from a dry reagent coating containedon a sample collection device. In still another aspect, the addingcomprises mixing the sample with a liquid comprising the IgM or thefragments thereof to form an amended mixture, the method furthercomprising the step of introducing the amended mixture into theimmunoassay device.

In another embodiment, the invention is to a device for performing animmunoassay of an analyte in a blood sample with reduced interferencefrom heterophile antibodies, comprising a housing, an electrochemicalimmunosensor, a conduit and a sample entry port, wherein said conduitpermits a blood sample to pass from the entry port to said immunosensor,and wherein at least one of said housing, said entry port, and saidconduit includes a dry reagent comprising non-human IgM or fragmentsthereof and IgG or fragments thereof at a weight ratio greater than0.004, e.g., greater than 0.02, or greater than 0.05.

In another embodiment, the invention is directed to methods for formingany of the above devices by depositing a liquid reagent cocktail in oneor more of the housing, the electrochemical immunosensor, the conduit orthe sample entry port, the reagent cocktail comprising the non-human IgMor the fragments thereof and optionally the IgG or the fragmentsthereof. The method further comprises the step of drying the reagentcocktail to form the dry reagent and assembling the device.

In some preferred embodiments of the methods and devices of theinvention, the analyte is TnI or BNP, and the dry reagent dissolves intothe sample to give an IgM concentration (or equivalent IgM fragmentconcentration) of from about 20 to about 200 μg/mL, e.g., 20 to about 60μg/mL, and an IgG concentration (or equivalent IgG fragmentconcentration) of from about 50 to about 5000 μg/mL, e.g., from about500 to about 1000 μg/mL.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other objectives, features and advantages of the presentinvention are described in the following detailed description of thespecific embodiments and are illustrated in the following Figures, inwhich:

FIG. 1 is an isometric top view of an immunosensor cartridge cover;

FIG. 2 is an isometric bottom view of an immunosensor cartridge cover;

FIG. 3 is a top view of the layout of a tape gasket for an immunosensorcartridge;

FIG. 4 is an isometric top view of an immunosensor cartridge base;

FIG. 5 is a schematic view of the layout of an immunosensor cartridge;

FIG. 6 is a schematic view of the fluid and air paths within animmunosensor cartridge, including sites for amending fluids with dryreagents;

FIG. 7 illustrates the principle of operation of an electrochemicalimmunosensor;

FIG. 8 is a side view of the construction of an electrochemicalimmunosensor with antibody-labeled particles not drawn to scale;

FIG. 9 is a top view of the mask design for the conductimetric andimmunosensor electrodes for an immunosensor cartridge;

FIG. 10 shows a table summarizing data for TnI and BNP cartridges withand without the heterophile antibody amelioration reagents;

FIG. 11 shows the TnI immunosensor and immuno-reference sensor responseversus time;

FIG. 12 shows the BNP immunosensor response and immuno-reference sensorversus time;

FIG. 13 is a schematic illustration of enzymatic regeneration of anelectroactive species;

FIG. 14 illustrates segment forming means;

FIG. 15 is a top view of the preferred embodiment of an immunosensorcartridge;

FIG. 16 is a schematic view of the fluidics of the preferred embodimentof an immunosensor cartridge;

FIG. 17(A-C) is a dose response plot for various troponin samples withheterophile antibodies at: (a) an immunosensor, (b) an associatedimmuno-reference sensor, and (c) the immunosensor signal subtracted fromthe immuno-reference sensor signal;

FIG. 18 illustrates the cartridge device with a slidable sealing elementfor closing the sample entry port in the closed position; and

FIG. 19 illustrates the cartridge device with a slidable sealing elementfor closing the sample entry port in the open position.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the reducing or eliminatinginterference caused by the presence of heterophile antibodies in thefollowing areas: (i) immunosensors, most notably in the context ofpoint-of-care testing, (ii) electrochemical immunoassays, (iii) the useof an immunosensor in conjunction with an immuno-reference sensor, (iv)whole blood immunoassays, (v) single-use cartridge based immunoassays,(vi) non-sequential immunoassays with only a single wash step, and (vii)dry reagent coatings including non-specific binding (NSB) inhibitors.However, as will be appreciated by those skilled in the art, the generalconcept is applicable to many immunoassay methods and platforms.

The present invention permits rapid in situ determinations of analytesusing a cartridge having an array of analyte sensors and means forsequentially presenting an amended sample to an immunosensor or analytearray. The cartridges are designed to be preferably operated with areading device, such as that disclosed in U.S. Pat. No. 5,096,669 toLauks et al., issued Mar. 17, 1992, or U.S. Pat. No. 7,419,821, issuedSep. 2, 2008, both of which are incorporated by reference herein intheir entireties. The present invention is best understood in thiscontext. Consequently, a suitable device and method of operation for apoint-of-care immunoassay system is first described, followed by how thesystem may be best adapted to reduce or eliminate heterophile antibodyinterference.

In one embodiment, the invention provides cartridges and methods oftheir use for processing liquid samples to determine the presence oramount of an analyte in the sample. The cartridges preferably contain ametering means, which permits an unmetered volume of sample to beintroduced, from which a metered amount is processed by the cartridgeand its associated reading apparatus. Thus, the physician or operator isrelieved of accurately measuring the volume of the sample prior tomeasurement saving time, effort, and increasing accuracy andreproducibility. The metering means, in one embodiment, comprises anelongated sample chamber bounded by a capillary stop and having alongits length an air entry point. Air pressure exerted at the air entrypoint drives a metered volume of the sample past the capillary stop. Themetered volume is predetermined by the volume of the sample chamberbetween the air entry point and the capillary stop.

The cartridge may have a closure device for sealing the sample port inan air-tight manner. This closure device is preferably slidable withrespect to the body of the cartridge and provides a shearing action thatdisplaces any excess sample located in the region of the port, reliablysealing a portion of the sample in the holding chamber between the entryport and the capillary stop. See, for example, Published US patentapplication US2005/0054078 A1, the entirety of which is incorporatedherein by reference. The cartridge may be sealed, for example, byslidably moving a sealing element over the surface of the cartridge in amanner that displaces excess fluid sample away from the sample orifice,seals a volume of the fluid sample within the internal fluid sampleholding chamber, and inhibits fluid sample from prematurely breakingthrough the internal capillary stop. The seal obtained by this slidableclosure device is preferably irreversible and prevents excess blood frombeing trapped in the cartridge because the closure device moves in theplane of the orifice through which blood enters the cartridge andprovides a shearing action that seals blood below the plane of the entryport, thereby moving excess blood, i.e., blood above the plane of theorifice, away from the entry port and optionally to a waste chamber.

One exemplary closure device is shown in FIG. 1 and comprises integratedelements 2, 3, 4 and 9 of cover 1. In this embodiment, closure device 2rotates about a hinge until hook 3 snaps shut blocking sample entry port4. An alternative to the closure device comprising integrated elements2, 3, 4 and 9 of cover 1 in FIG. 1 is shown as a separate slidableelement 200 in FIGS. 18 and 19. FIGS. 18 and 19 show a cartridge devicecomprising a modified version of the cover of FIG. 1 attached to a basesimilar to the base in FIG. 4 with intervening adhesive layer 21 shownin FIG. 3 along with the separate slidable closure element 200. FIG. 19shows the closure device 200 in the open position, where the sampleentry port 4 can receive a sample, e.g., blood. FIG. 18 shows theclosure device 200 in the closed position where it seals the sampleentry port in an air-tight manner. In operation, element 200 is manuallyactuated from the open to the closed position after the sample, e.g.,blood, has been added to the entry port and it enters the holdingchamber 34. In the embodiment shown, any excess blood in the region ofthe entry port is moved into an overflow chamber 201 or an adjacentretaining region or cavity. This chamber or region may include afluid-absorbing pad or material to retain the excess sample, e.g.,blood.

The sample entry port 4 may be an orifice that is circular, as shown inFIG. 19, or oval and the diameter of the orifice is generally in therange 0.2-5 mm, preferably 1-2 mm, or having a perimeter of 1-15 mm foran oval. The region around the orifice may be selected to be hydrophobicor hydrophilic to control the drop-shape of the applied sample topromote entry into the entry port. One advantage of the closure deviceshown in FIGS. 18 and 19 is that it prevents the sample from beingpushed beyond the capillary stop element 25 at the end of the holdingchamber 34. The presence of a small amount of sample, e.g., blood,beyond the capillary stop is not significant for tests that measure bulkconcentration of an analyte and thus do not depend on sample volume.However, for immunoassay applications where metering of the sample isgenerally advantageous the sealing element improves metering accuracy ofthe device and assures the assayed segment of sample is appropriatelypositioned with respect to the immunosensor when the analyzer actuatesthe sample within the cartridge conduits.

In operation, when the sample, e.g., blood, is added to the cartridge itmoves to the capillary stop. Thus sufficient sample for the assay ispresent when the region from the capillary stop to the sample entryport, i.e., the holding chamber 34, contains the sample. During theprocess of filling the holding chamber some sample may remain above theplane of the orifice of the entry port. When the sealing element ismoved from the opened to closed position, any sample that is above theentry port is sheared away without trapping additional sample in the actof closure, thus ensuring that the sample does not move beyond capillarystop 25. In a preferred embodiment, sealing element 200 is positionedwithin a few thousandths of an inch above the surface of the tape gasket21 of FIG. 3. The entry port is sealed by the subsequent lowering of thesurface of 200 to the adhesive tape gasket when it engages lockingfeatures 212 and 213. Since the tape is essentially incompressible andthe orifice has a small diameter, any inadvertent pressure applied tothe sealing element by the user will not cause the sample to move beyondthe capillary stop.

In certain cartridge embodiments that use several drops of sample, it isdesirable that no bubbles form in the holding chamber as this can affectthe assay. Accordingly, a reliable means for introducing more than onedrop of sample, e.g., blood, into the holding chamber 34 withoutentraining bubbles has been developed. The sample entry port can bedesigned to receive multiple drops of sample without successive dropscausing trapped bubbles to form in the holding chamber 34 by firsttreating the holding chamber with a Corona and/or a reagent cocktail.

The use of Corona treatments on disposable medical devices is well knownin the art and is an effective way to increase the surface activity ofvirtually any material, e.g., metallized surfaces, foils, paper,paperboard stock, or plastics such as polyethylene, polypropylene,nylon, vinyl, PVC, and PET. This treatment makes them more receptive toinks, coatings, and adhesives. In practice the material being treated isexposed to an electrical discharge, or “corona.” Oxygen molecules in thedischarge area break into atoms and bond to molecules in the materialbeing treated, resulting in a chemically activated surface. Suitableequipment for corona treatments is commercially available (e.g. CorotecCorp., Farmington, Conn.). The process variables include the amount ofpower required to treat the material, the material speed, the width, thenumber of sides to be treated, and the responsiveness of a particularmaterial to corona treatment, which variables can be determined by askilled operator. The typical place to install a corona treatment isin-line with the printing, coating, or laminating process. Anothercommon installation is directly on a blown film or cast film extrudersince fresh material is more receptive to corona treatment.

As described above, the sandwich immunoassay format is the most widelyused immunoassay method and it is also the preferred format in theanalysis device, e.g., cartridge, discussed herein. In this embodiment,one antibody (the immobilized antibody) is bound to a solid support orimmunosensor, and a second antibody (the signal antibody) isconjugated/bound to a signal-generating reagent such as an enzyme, e.g.,alkaline phosphatase. The signal-generating reagent (e.g., signalantibody) may be part of a dry reagent coating in the analysis device,as described below, and preferably dissolves into the biological samplebefore the sample reaches the immunosensor. After washing away thesample and non-specifically bound reagents, the amount ofsignal-generating reagent (e.g., signal antibody) remaining on the solidsupport should in principle be proportional to the amount of analyte inthe sample. However, one limitation of the assay configuration is thesusceptibility to interference(s) caused by heterophile antibodies thatmay be present in the biological sample. Specifically, endogenousantibodies capable of binding to one or more of the assay reagents posethe potential to generate erroneous test results by cross-linking thereagents (false-positive) or sequestering the reagents (false-negative).

While many heterophile interferences are mitigated by the addition ofmouse IgG, it has surprisingly been found that IgG is ineffective formitigating heterophile interference for certain samples. In thesesamples, the system recognizes a sample error even in the presence ofIgG and, as a result, an algorithm in the device causes the device tosuppress the reporting of the erroneous result. This was found for somesamples even in the presence of significant amounts of IgG.

It has now been discovered that these systems yield accurate resultsonly in the additional presence of IgM class immunoglobulins orfragments thereof isolated from animal species. As used herein, the term“fragment” refers to any epitope-bearing fragment derived from thespecified molecule. Thus, an IgM fragment may comprise, for example, aF(ab′)2 fragment, a Fab fragment or a Fc fragment, which areepitope-bearing fragments of the IgM molecule. Further, by “IgM orfragments thereof” it is meant IgM alone, IgM fragments alone (i.e., oneor more of F(ab′)2 fragments, Fab fragments and/or Fc fragments of IgM),or a combination of IgM and IgM fragments.

In a preferred embodiment, the IgM or fragments thereof are incorporatedinto a dry reagent coating, which in some embodiments may be the samedry reagent coating that contains the signal-generating reagent (e.g.,signal antibody). Thus, in one embodiment, the analysis device includesa dry reagent coating that comprises either or both: (a) a componentsuitable for ameliorating the effect of heterophile antibodies, e.g.,IgM or fragments thereof and preferably IgG or fragments thereof, and/or(b) a signal antibody. The dry reagent coating may be formed from areagent cocktail, which also preferably comprises either or both: (a) acomponent suitable for ameliorating the effect of heterophileantibodies, e.g., IgM or fragments thereof and preferably IgG orfragments thereof, and/or (b) a signal antibody. The surface on whichthe reagent cocktail is to be deposited preferably is first Coronatreated to provide charged surface groups that will promote spreading ofthe printed cocktail.

In general, the reagent cocktail used to form the dry reagent coatingmay further comprise a water-soluble protein, an amino acid, apolyether, a polymer containing hydroxyl groups, a sugar orcarbohydrate, a salt and optionally a dye molecule. One or more of eachcomponent can be used. In one embodiment, the cocktail contains bovineserum albumin (BSA), glycine, salt, methoxypolyethylene glycol, sucroseand optionally bromophenol blue to provide color that aids visualizingthe printing process. In one embodiment, from 1 to 20 μL of cocktail isprinted onto the desired surface, e.g., within the holding chamber orother conduit, of the analysis device and allowed to air dry (or heat isdried) before being assembled with its cover.

In another embodiment, the test cartridge may comprise a plurality ofdry reagent coatings (in which case the coatings may be respectivelyreferred to as a first reagent coating, a second reagent coating, etc.,in order to distinguish them). For example, the IgM or fragments thereofmay be included in a first reagent coating, which, for example, may beadjacent to a second reagent coating that contains the signal generatingelement, e.g., signal antibody. In this aspect, the second reagentcoating may be located upstream or downstream of the first reagentcoating, although it is preferable for the reagent coating that containsthe signal antibody to be located downstream of the reagent coating thatcontains the component for inhibiting heterophile antibody interference.In a preferred embodiment, the holding chamber is coated with a firstreagent coating that comprises IgG or fragments thereof and IgM orfragments thereof. In this aspect, a second reagent coating comprisingthe signal antibody preferably is located downstream of the holdingchamber, e.g., immediately upstream of the immunosensor.

In still other embodiments, the IgM or fragments and thereof may not bepart of the analysis device, e.g., cartridge. For example, a firstreagent coating comprising IgM or fragments thereof and preferably IgGor fragments thereof may be incorporated in a sample collection device,e.g., capillary or syringe. For example, the first reagent coating maybe formed on an interior wall of the capillary or syringe.

In another embodiment, the component(s) for ameliorating heterophileinterference may be contained in solution and mixed with the biologicalsample, e.g., blood, and the resulting amended sample is introduced intothe analysis device, e.g., cartridge. In one embodiment, for example, ablood sample may be mixed with a liquid comprising IgM or fragmentsthereof (and preferably IgG or fragments thereof) to form an IgM amendedsample, which is then introduced into the analysis device, e.g.,cartridge. In another aspect, the device includes a pouch therein thatcontains a liquid comprising IgM or fragments thereof (and preferablyIgG or fragments thereof), which is mixed with a biological sample inthe device and then processed substantially as described herein to forma sandwich assay for analyte detection.

In another embodiment, electrowetting is employed to mix a first liquidcomprising IgM or fragments thereof and preferably IgG or fragmentsthereof with a liquid biological sample such as blood. In thisembodiment, an apparatus may be provided for manipulating droplets. Theapparatus, for example, may have a single-sided electrode design inwhich all conductive elements are contained on one surface on whichdroplets are manipulated. An additional surface can be provided parallelwith the first surface for the purpose of containing the droplets to bemanipulated. Droplets are manipulated by performing electrowetting-basedtechniques in which electrodes contained on or embedded in the firstsurface are sequentially energized and de-energized in a controlledmanner. The apparatus may allow for a number of droplet manipulationprocesses, including merging and mixing two droplets together, splittinga droplet into two or more droplets, sampling a continuous liquid flowby forming from the flow individually controllable droplets, anditerative binary or digital mixing of droplets to obtain a desiredmixing ratio. In this manner, droplets of the first liquid comprisingIgM or fragments thereof may be carefully and controllably merged andmixed with the liquid biological sample, e.g., blood. See, e.g., U.S.Pat. No. 6,911,132, the entirety of which is incorporated herein byreference.

While the present invention is broadly applicable to immunoassaysystems, it is best understood in the context of the i-STAT™ immunoassaysystem (Abbott Point of Care Inc., Princeton, N.J.), as described injointly owned pending and issued patents cited above. In someembodiments, the system employs an immuno-reference sensor (See US2006/0160164 A1, incorporated herein by reference in its entirety) forpurposes of assessing the degree of NSB occurring during an assay. NSBmay arise due to inadequate washing or due to the presence ofinterferences. The net signal from the assay is comprised of thespecific signal arising from the analyte immunosensor corrected bysubtracting the non-specific signal arising from the immuno-referencesensor. The amount of signal at the immuno-reference sensor is subjectto limits defined by a quality control algorithm.

In one embodiment, the present invention improves the resistance of thei-STAT immunoassay format to interference by endogenous antibodies,however it is equally applicable to the standard ELISA format.Specifically, the invention involves the use of IgM classimmunoglobulins which have been found to substantially reduce theinterference caused by heterophile antibodies in certain test specimens.

As indicated above, it has now been discovered that amending a samplewith IgM class immunoglobulins (or fragments thereof) preferably incombination with IgG class immunoglobulins (or fragments thereof)isolated from animal species results in reduced or eliminatedheterophile antibody interference. In experiments, mouse immunoglobulinM (IgM, Sigma-Aldrich) was added to the sample conditioning printcocktail used in the i-STAT™ immunoassay format. Then known patientplasma samples that could not previously be reliably analyzed in thefield due to interference from heterophile antibodies were tested. Asindicated above, it should be noted that the i-STAT™ system did notpreviously report inaccurate results for these samples, as the systemincludes a failsafe algorithm that detects spurious signals at theimmuno-reference sensor, alerts the user with an error code, andsuppresses the result from being displayed. This is an example of onepart of a quality system required for reliable point-of-care testing.

Surprisingly, when the new mouse IgM modified cartridges were tested andthe results compared with the conventional cartridges, the resultsobtained demonstrate that the previously problematic plasma samples cannow be analyzed accurately when IgM-modified cartridges are employed.

The data in FIG. 10 summarizes test results in which the efficacy ofmurine IgM in measurement of two cardiac markers, cardiac troponin I(cTnI) and brain-type natriuretic peptide (BNP), in two respectivesamples (Samples 1 and 2) exhibiting known heterophile antibodyinterference was determined. In FIG. 10, the “mean result” refers to theanalyte concentration computed from a net differential signal (signal atanalyte sensor minus signal at reference sensor), “number of cartridges”refers to the number of assays performed, and “number of errors” refersto the number of results suppressed due to excessive NSB at thereference sensor among the assays performed on the sample for a givenformat.

In the cTnI tests described in FIG. 10, a set of nine tests wereperformed on a blood sample (Sample 1) with the new IgM modifiedcartridges (MOD). The average result was 0.23 ng/mL and there were noidentified system errors, whereas for the standard (STD) devices allnine cartridges gave an error reported by the system, the average resultbeing a meaningless “negative” concentration (−0.21 ng/mL). (The originof “negative” values in this context is explained below.) Thus, with theMOD cartridges of the invention, all nine samples passed the internalsystem quality checks and yielded a meaningful result, whereas the STDcartridges all recognized an error associated with the sample andsuppressed reporting a result.

In the BNP test set, a series of three tests were performed on a secondsample (Sample 2) with the new IgM MOD cartridges and with the STDcartridges. For the MOD cartridges of the invention, the average resultwas −27 pg/mL and there were no identified system errors, whereas forthe STD devices, all three tests gave an error reported by the system,the average result being −157 pg/mL. Again, the origin of “negative”values in this context is explained below.

To provide further insight, FIGS. 11 and 12 illustrate the actual rawtransient electrochemical immunosensor responses and theimmuno-reference sensor response associated with the measurementssummarized for the two samples (Samples 1 and 2) containing heterophileinterferences in FIG. 10.

Sample 1 (FIGS. 10 and 11) arose in clinical practice and was determinedto be positive for cTnI. The test cartridge employed was an i-STAT™Cardiac Troponin I cartridge and contained a sensor for Cardiac TroponinI (solid lines, FIG. 11) and an immuno-reference sensor used to assessthe degree of NSB of the anti-cTnI conjugate reagent. See US2006/0160164 A1.

FIG. 11 shows the change in electrochemical immunosensor responses for asample containing heterophile antibody activity upon incorporation ofmurine IgM. The solid lines indicate the amperometric response at animmunosensor for cTnI bearing anti-cTnI antibodies before (light solidline) and after (heavy solid line) treatment with IgM. Dashed linesindicate the amperometric response at an immuno-reference sensor forhuman serum albumin (HSA) bearing anti-HSA antibodies before (lightdashed line) and after (heavy dashed line) treatment with IgM. Arrowsindicate the change in response upon addition of IgM.

NSB can arise due to a problem with the wash step or due to the presenceof interference(s). The immuno-reference sensor employed was comprisedof an electrode bearing a coating of anti-HSA antibody labeledmicroparticles and becomes coated with HSA upon exposure to sample (notethat HSA arises naturally from the sample). Inspection of FIG. 11indicates that the signal arising from the cTnI immunosensor (solidlines) increases upon addition of murine IgM (heavy line). Thus, in theabsence of IgM, the troponin concentration was underestimated due to thepresence of the heterophile interference(s). This suggests that thesample contained interferents capable of binding to one or more of theimmunoreagents (anti-cTnI antibody) immobilized over the immunosensor oranti-HSA conjugated to the enzyme alkaline phosphatase (ALP) therebydecreasing their availability for interaction with the analyte. Alsoevident from FIG. 11 is that before addition of murine IgM (lightlines), the immuno-reference sensor (dashed lines) exhibited asignificant signal that diminished markedly upon addition of murine IgM.Thus, the IgM acts to mitigate an interferent that is capable ofcross-linking the anti-cTnI conjugate reagent (containing animalanti-cTnI antibodies) with the anti-HSA reagent (on the immuno-referencesensor).

It is notable that while traditional sandwich assays would yielderroneous results in these cases, the i-STAT system assay yields anerror code owing to error detection algorithms that require the signalfrom the reference sensor to be below a critical value. This is muchsuperior, since it is clinically highly desirable to not report a resultas opposed to reporting one in error. In this way the quality andintegrity of the analytical system is maintained.

In general, commercial assays do not include such a referencemeasurement, e.g., one based on an immuno-reference sensor, but relyinstead on the absence or adequate neutralization of interferents in thesamples measured. In the absence of this reference measurement theSample 1 (FIG. 10) would have reported 0.00 ng/mL. Note that the actualvalue of −0.21 arises from subtracting the relatively largeimmuno-reference sensor current from the smaller analyte sensor current.This is obviously non-physical (a meaningless value or result) and isreported as zero. The true sample value is actually positive fortroponin (0.23 ng/mL) as obtained in the presence of IgM. Note also thatSample 1 had a very low cTnI concentration. Thus, the actualimmunosensor (IS) signal would be expected to be low. Further, it is notunexpected that the signal at the immuno-reference sensor (IRS) may beslightly higher than the IS signal resulting in a negative value sincethe IRS signal is subtracted from the IS signal. Here, we areparticularly interested in assessing the effect of heterophileantibodies on samples with intrinsically low analyte concentrations,where the relative effect of the former on the latter can besignificant. In samples where the actual analyte concentration is at thehigher end of the range, the relative effect of heterophile antibodieswill generally be less pronounced. Further detail on additional studiesof troponin is shown in FIG. 17(A-C) which provides a dose response plotfor various troponin samples with heterophile antibodies at: (a) animmunosensor, (b) an associated immuno-reference sensor, and (c) theimmunosensor signal subtracted from the immuno-reference sensor signal.A detailed description of FIG. 17 is found in Example 5 below.

FIG. 12 shows changes in electrochemical BNP immunosensor response for asample containing heterophile antibody activity upon incorporation ofmurine IgM. Solid lines indicate the amperometric response at animmunosensor for BNP bearing anti-BNP antibodies before (light solidline) and after (heavy solid line) treatment with IgM. Dashed linesindicate the amperometric response at an immunosensor for human serumalbumin (HSA) bearing anti-HSA antibodies before (light dashed line) andafter (heavy dashed line) treatment with IgM. Arrows indicate the changein response upon addition of IgM.

Sample 2 (FIGS. 10 and 12) arose in a population of normal nominallyhealthy individuals and in fact has a low BNP concentration. The testcartridge (i-STAT Brain-type Natriuretic Peptide, BNP) contained animmunosensor for BNP (solid lines, FIG. 12) and an immuno-referencesensor used to assess the degree of NSB of the anti-BNP conjugatereagent. As indicated above, NSB can arise due to inadequacy of the washstep or due to the presence of interference(s). The immuno-referencesensor is comprised of an electrode bearing a coating of anti-HSA (humanserum albumin) antibody labeled microparticles and becomes coated withHSA upon exposure to sample (HSA arises naturally from the sample).Inspection of FIG. 12 indicates that the signal arising from the BNPimmunosensor (solid lines) decreases upon addition of murine IgM (heavyline). Thus, in the absence of IgM, the BNP concentration isoverestimated at this sensor due to the presence of the heterophileinterference. Also evident in FIG. 12 is that before addition of murineIgM (light lines), the immuno-reference sensor (dashed lines) exhibits asignificant signal that diminishes markedly upon addition of murine IgM(heavy lines). Thus the IgM acts to mitigate an interferent that iscapable of cross-linking the anti-BNP conjugate reagent (containinganimal anti-BNP antibodies) and either the anti-BNP or the anti-HSAreagent (on the analyte and immuno-reference sensor surfaces).

In the absence of the immuno-reference sensor or IgM, the signalobserved from the BNP sensor (light solid line, FIG. 12) wouldcorrespond to a falsely elevated result. The interference-induced signalon the immuno-reference sensor (light dashed line, FIG. 12) allowed the(pre-IgM mitigation) result to be suppressed in the i-STAT™ assayformat. Thus, inclusion of IgM allows a correct result (0 pg/mL BNP) tobe reported. The occurrence of apparently negative results follows thesame type of explanation as for the cTnI example above.

With regard to the new dry reagent including IgM that is optionallyprinted into the cartridge, as described above, the reagent preferablyis formulated as an aqueous solution containing interference-eliminatingreagents such as murine and caprine IgG and murine IgM. As discussedabove, upon introduction of a biological sample, e.g., blood, the samplepreferably mixes with the reagent in a first step of the assay. Thereagent may also include inorganic salts and surfactants to optimizeassay performance with respect to chemical and fluidic attributes. Otheroptional additives include heparin to ensure adequate anticoagulationand dyes for visualization of the location of the reagent afterprinting. Also optionally present are stabilizers such as sodium azidefor inhibition of microbial growth and a mixture of lactitol anddiethylaminoethyl-dextran (Applied Enzyme Technologies Ltd., MonmouthHouse, Mamhilad Park, Pontypool, NP4 OHZ UK) for stabilization ofproteins.

For reduction of heterophile antibody interference, murine IgM may, forexample, be incorporated in such an amount that the sample is dosed toconcentrations of about 10 μg/mL to about 100 μg/mL with a preferredrange of 25 to 40 μm/mL IgM. The liquid reagent preferably is preparedat concentrations ranging from 1 wt. % solids to 30 wt. % solids withthe preferred concentration in the 5 to 7 wt. % solids range. Oncedeposited in the device, the deposited reagent may, for example, bedried for 30 to 60 minutes in a stream of warm air. In one embodiment,the reagent is printed in the sample inlet of the device using anautomated printing instrument and dried to form an IgM-containingreagent coating layer.

In a preferred embodiment as implemented in the Cardiac Troponin Iimmunoassay, the base print cocktail is prepared as follows for a 1liter (L) batch: Protein stabilization solution (PSS, AET Ltd., 50%solids, 100.0 g) is added to 200-250 mL of an aqueous solution of sodiumchloride (8.00 g) and sodium azide (0.500 g) and the resulting solutionis transferred to a 1 L volumetric flask. A solution of murine IgG isprepared by adding murine IgG (0.9 g) to 75 mL of deionized water andstirred for 15-60 minutes until dissolution is complete. An equallyconcentrated solution of caprine IgG is prepared in an identical mannerand both solutions are filtered through a 1.2 μM filter. Murine IgM isacquired as a liquid from the supplier (for example, Sigma-Aldrich). Theprotein concentrations of each of the three immunoglobulin (Ig) stocksolutions are measured spectrophotometrically at 280 nm. The masses ofthese Ig solutions required to provide murine IgG (0.75 g), caprine IgG(0.75 g) and murine IgM (25 mg) are calculated and these amounts areadded to the printing solution. A solution of diethylaminoethyl-dextran(DEAE-dextran) is prepared by adding DEAE-dextran (2.5 g) to 50-100 mLof deionized water and stirred for 30 minutes. The DEAE-dextran solutionis added to the printing solution. To this is added sodium heparin(10,000 IU/mL, 3.00 mL), Tween-20 (3.00 g) and a 5% (w/v) aqueoussolution of Rhodamine (200 μL). The resulting solution is diluted to1.000 L with deionized water and stored in a freezer or refrigeratoruntil use.

Printing of these and similar fluids to form a dry reagent coating onthe cartridge component is preferably automated and based on amicrodispensing system, including a camera and computer system to aligncomponents, as disclosed in U.S. Pat. No. 5,554,339. In this patent, thewafer chuck is replaced by a track for feeding the plastic cartridgebases to the dispensing head. The track presents the bases to the headin a predetermined orientation to ensure consistent positionaldispensing.

Wafer-level microfabrication of a preferred embodiment of theimmunosensor is as follows. The base electrode (94 of FIG. 9) comprisesa square array of 7 μm gold disks on 15 μm centers. The array covers acircular region approximately 600 μm in diameter, and is achieved byphoto-patterning a thin layer of polyimide of thickness 0.35 μm over asubstrate made from a series of layers comprising Si/SiO₂/TiW/Au. Thearray of 7 μm microelectrodes affords high collection efficiency ofelectroactive species with a reduced contribution from anyelectrochemical background current associated with the capacitance ofthe exposed metal. The inclusion of a PVA layer over the metalsignificantly enhances the reduction of background currents.

The porous PVA layer is prepared by spin-coating an aqueous mixture ofPVA plus a stilbizonium photoactive, cross-linking agent over themicroelectrodes on the wafer. The spin-coating mixture optionallyincludes bovine serum albumin (BSA). It is then photo-patterned to coveronly the region above and around the arrays and preferably has athickness of about 0.6 μm.

The general concept of differential measurement is known in theelectrochemical and sensing arts. A novel means for reducing interferingsignals in an electrochemical immunosensing systems is now described.However, while it is described for pairs of amperometric electrochemicalsensors it is of equal utility in other electrochemical sensing systemsincluding potentiometric sensors, field effect transistor sensors andconductimetric sensors. It is also applicable to optical sensors, e.g.,evanescent wave sensors and optical wave guides, and also other types ofsensing including acoustic wave and thermometric sensing and the like.Ideally, the signal from an immunosensor (IS) is derived solely from theformation of a sandwich comprising an immobilized antibody (Ab1), theanalyte, and a signal antibody (Ab2) that is labeled, wherein the label(e.g., an enzyme) reacts with a substrate (S) to form a detectableproduct (P) as shown below in scheme (1).

Surface−Ab1−analyte−Ab2−enzyme; enzyme+S→P   (1)

It is known that some of the signal antibody (Ab2) may bindnon-specifically to the surface, as shown below in schemes (2) and (3),and might not be washed away completely from the region of theimmunosensor (up to approx. 100 microns away) during the washing stepgiving rise to a portion of the total detected product that is not afunction of the surface-Ab1-analyte-Ab2-enzyme immunoassay sandwichstructure, thereby creating an interfering signal.

Surface−Ab2−enzyme; enzyme+S→P   (2)

Surface−analyte−Ab2−enzyme; enzyme+S→P   (3)

As indicated above, a second immunosensor optionally may be placed inthe cartridge that acts as an immuno-reference sensor (IRS) and givesthe same (or a predictably related) degree of NSB as occurs on theprimary immunosensor. Interference can be reduced by subtracting thesignal of this immuno-reference sensor from that of the primaryimmunosensor, i.e., the NSB component of the signal is removed,improving the performance of the assay, as shown in scheme (4) below.This correction may optionally include the subtraction or addition of anadditional offset value.

Corrected signal=IS−IRS   (4)

The immuno-reference sensor is preferably the same in all significantrespects (e.g., dimensions, porous screening layer, latex particlecoating, and metal electrode composition) as the primary immunosensorexcept that the capture antibody for the analyte (for instance, cTnI) isreplaced by an antibody to a plasma protein that naturally occurs insamples (both normal and pathological) at a high concentration. Theimmunosensor and reference immunosensor may be fabricated as adjacentstructures 94 and 96, respectively, on a silicon chip as shown in FIG.9. While the preferred embodiment is described for troponin I and BNPassays, this structure is also useful for other cardiac marker assaysincluding, for example, troponin T, creatine kinase MB, procalcitonin,proBNP, myoglobin and the like, plus other sandwich assays used inclinical diagnostics, e.g., PSA and TSH.

Examples of suitable antibodies that bind to plasma proteins includeantibodies to human serum albumin, fibrinogen and IgG fc region, withalbumin being preferred. However, any native protein or blood componentthat occurs at a concentration of greater than about 100 ng/mL can beused if an appropriate antibody is available. The protein should,however, be present in sufficient amounts to coat the sensor quicklycompared to the time needed to perform the analyte assay. In a preferredembodiment, the protein is present in a blood sample at a concentrationsufficient to bind more than 50% of the available antibody on thereference immunosensor within about 100 seconds of contacting a bloodsample. In general the second immobilized antibody has an affinityconstant of about 1×10⁻⁷ to about 1×10⁻¹⁵ M. For example, an antibody toalbumin having an affinity constant of about 1×10⁻¹⁰ M is preferred, dueto the high molar concentration of albumin in blood samples, which isabout 1×10⁻⁴ M.

It has been found that providing a surface that is covered by nativealbumin derived from the sample significantly reduces the binding ofother proteins and cellular materials that may be present. This methodis generally superior to conventional immunoassays that use conventionalblocking agents to minimize NSB because these agents must typically bedried down and remain stable for months or years before use, duringwhich time they may degrade, creating a stickier surface than desiredand resulting in NSB that rises with age. In contrast, the methoddescribed here provides a fresh surface at the time of use.

An immunosensor for cardiac troponin I (cTnI) with areference-immunosensor for performing differential measurement to reducethe effect of NSB is described next. Carboxylate-modified latexmicroparticles (supplied by Bangs Laboratories Inc. or SeradynMicroparticles Inc.) coated with anti-cTnI and anti-HSA are bothprepared by the same method. The particles are first buffer exchanged bycentrifugation, followed by addition of the antibody, which is allowedto passively adsorb onto the particles. The carboxyl groups on theparticles are then activated with EDAC in MES buffer at pH 6.2, to formamide bonds to the antibodies. Any bead aggregates are removed bycentrifugation and the finished beads are stored frozen.

It was found that for the anti-human serum albumin (HSA) antibody,saturation coverage of the latex beads results in about a 7% increase inbead mass. Coated beads were prepared using covalent attachment from amixture comprising 7 mg of anti-HSA and 100 mg of beads. Using thispreparation a droplet of about 0.4 nL, comprising about 1% solids indeionized water, was microdispensed (using the method and apparatus ofU.S. Pat. No. 5,554,339, incorporated herein by reference in itsentirety) onto a photo-patterned porous polyvinyl alcohol permselectivelayer covering sensor 96, and allowed to dry. The dried particlesadhered to the porous layer and substantially prevented theirdissolution in the blood sample or the washing fluid.

For the troponin antibody, saturation coverage of the latex bead surfaceresulted in a mass increase in the beads of about 10%. Thus by adding 10mg of anti-TnI to 100 mg of beads along with the coupling reagent,saturation coverage was achieved. These beads were then microdispensedonto sensor 94.

In another embodiment, immunosensor 94 is coated with beads having botha plasma protein antibody, e.g., anti-HSA, and the analyte antibody,e.g., anti-cTnI. Latex beads made with the about 2 mg or less ofanti-HSA per 100 mg of beads and then saturation-coated with anti-cTnIprovide superior NSB properties at the immunosensor. It has been foundthat the slope (signal versus analyte concentration) of the troponinassay is not materially affected because there is sufficient anti-cTnIon the bead to capture the available analyte (antigen). By determiningthe bead saturation concentration for different antibodies, and theslope of an immunosensor having beads with only the antibody to thetarget analyte, appropriate ratios of antibody combinations can bedetermined for beads having antibodies to both a given analyte and aplasma protein.

An important aspect of immunosensors having a reference immunosensor isthe “humanizing” of the surface created by a layer of plasma protein,preferably the HSA/anti-HSA combination. This appears to make the beadsless prone to NSB of the antibody-enzyme conjugate. It also seems toreduce bead variability. Without being bound by theory, it appears thatas the sensors are covered by the sample they are rapidly coated withnative albumin due to the anti-HSA surface. This gives superior resultscompared to conventional blocking materials, which are dried down inmanufacturing and re-hydrated typically after a long period in storage.Another advantage to “humanizing” the sensor surface is that it providesan extra mode of resistance to human anti-mouse antibodies (HAMA) andother heterophile antibody interferences. The effects of HAMA onimmunoassays are well known.

Another use of the immuno-reference sensor, which may be employed in thedevices and methods of the invention, is to monitor the wash efficiencyobtained during the analytical cycle. As stated above, one source ofbackground noise is the small amount of enzyme conjugate still insolution, or non-specifically absorbed on the sensor and not removed bythe washing step. This aspect of the invention relates to performing anefficient washing step using a small volume of washing fluid, byintroducing air segments as mentioned in Example 2.

In operation of the preferred embodiment, which is an amperometricelectrochemical system, the currents associated with oxidation ofp-aminophenol at immunosensor 94 and immuno-reference sensor 96 arisingfrom the activity of ALP, are recorded by the analyzer. The potentialsat the immunosensor and immuno-reference sensor are poised at the samevalue with respect to a silver-silver chloride reference electrode. Toremove the effect of interference, the analyzer subtracts the signal ofthe immuno-reference sensor from that of the immunosensor according toequation (4) above. Where there is a characteristic constant offsetbetween the two sensors, this also is subtracted. It will be recognizedthat it is not necessary for the immuno-reference sensor to have all thesame non-specific properties as the immunosensor, only that it beconsistently proportional in both the wash and NSB parts of the assay.An algorithm embedded in the analyzer can account for any otheressentially constant difference between the two sensors.

Use of a differential combination of immunosensor and immuno-referencesensor, rather than an immunosensor alone, provides the followingimprovement to the assay. In a preferred embodiment the cartridge designprovides dry reagent that yields about 4-5 billion enzyme conjugatemolecules dissolved into about a 10 μL blood sample. At the end of thebinding and wash steps the number of enzyme molecules at the sensor isabout 70,000. In experiments with the preferred embodiment there were,on average, about 200,000 (±about 150,000) enzyme molecules on theimmunosensor and the reference immunosensor as non-specifically boundbackground. Using a differential measurement with the immuno-referencesensor, about 65% of the uncertainty was removed, significantlyimproving the performance of the assay. While other embodiments may haveother degrees of improvement, the basis for the overall improvement inassay performance remains.

An additional use of the optional immuno-reference sensor is to detectanomalous sample conditions, such as improperly anti-coagulated sampleswhich deposit material throughout the conduits and cause increasedcurrents to be measured at both the immunosensor and theimmuno-reference sensor. This effect is associated with bothnon-specifically adsorbed enzyme and enzyme remaining in the thin layerof wash fluid over the sensor during the measurement step.

Another use of the optional immuno-reference sensor is to correctsignals for washing efficiency. In certain embodiments the level ofsignal at an immunosensor depends on the extent of washing. For example,longer washing with more fluid/air segment transitions can give a lowersignal level due to a portion of the specifically bound conjugate beingwashed away. While this may be a relatively small effect, e.g., lessthan 5%, correction can improve the overall performance of the assay.Correction may be achieved based on the relative signals at the sensors,or in conjunction with a conductivity sensor located in the conduitadjacent to the sensors, acting as a sensor for detecting and countingthe number of air segment/fluid transitions. This provides the input foran algorithmic correction means embedded in the analyzer.

In another embodiment of the reference immunosensor with an endogenousprotein, e.g., HSA, it is possible to achieve the same goal by having animmuno-reference sensor coated with antibody to an exogenous protein,e.g., bovine serum albumin (BSA). In this case the step of dissolving aportion of the BSA in the sample, provided as an additional reagent,prior to contacting the sensors is needed. This dissolution step can bedone with BSA as a dry reagent in the sample holding chamber of thecartridge, or in an external collection device, e.g., a BSA-coatedsyringe. This approach offers certain advantages, for example theprotein may be selected for surface charge, specific surface groups,degree of glycosylation and the like. These properties may notnecessarily be present in the available selection of endogenousproteins.

In addition to salts, other reagents can improve whole-blood precisionin an immunoassay. These reagents should be presented to the bloodsample in a way that promotes rapid dissolution. Support matricesincluding cellulose, polyvinyl alcohol and gelatin (or mixtures thereof)that are coated on to the wall of the blood-holding chamber (or anotherconduit) promote rapid dissolution, e.g., greater than 90% complete inless than 15 seconds.

In addition to the inclusion of IgM or fragments thereof, other optionaladditives may be included in the cartridge or used in conjunction withthe assay. The anticoagulant heparin can be added to improve performancein cases where the sample was not collected in a heparinized tube or wasnot properly mixed in a heparinized tube. Enough heparin is added sothat fresh unheparinized blood will remain uncoagulated during the assaycycle of the cartridge, typically in the range of 2 to 20 minutes. Goatand mouse IgG can by added to combat heterophile antibody problems wellknown in the immunoassay art. Proclin, DEAE dextran, Tris buffer andlactitol can be added as reagent stabilizers. Tween 20 can be added toreduce binding of proteins to the plastic, which is the preferredmaterial for the cartridge. It also allows the reagents to coat theplastic surface more evenly and acts as an impurity that minimizes thecrystallization of sugars, such as lactitol, so that they remain aglass. Sodium azide may be added to inhibit bacterial growth.

A cartridge of the present invention has the advantage that the sampleand a second fluid can contact the sensor array at different timesduring an assay sequence. The sample and the second fluid may also beindependently amended with other reagents or compounds present initiallyas dry coatings within the respective conduits. Controlled motion of theliquids within the cartridge further permits more than one substance tobe amended into each liquid whenever the sample or fluid is moved to anew region of the conduit. In this way, provision is made for multipleamendments to each fluid, greatly extending the complexity of automatedassays that can be performed, and therefore enhancing the utility of thepresent invention.

In a disposable cartridge, the amount of liquid contained is preferablykept small to minimize cost and size. Therefore, in the presentinvention, segments within the conduits may also be used to assist incleaning and rinsing the conduits by passing the air-liquid interface ofa segment over the sensor array or other region to be rinsed at leastonce. It has been found that more efficient rinsing, using less fluid,is achieved by this method compared to continuous rinsing by a largervolume of fluid.

Restrictions within the conduits serve several purposes in the presentinvention. A capillary stop located between the sample holding chamberand first conduit is used to facilitate sample metering in the holdingchamber by preventing displacement of the sample in the holding chamberuntil sufficient pressure is applied to overcome the resistance of thecapillary stop. A restriction within the second conduit is used todivert wash fluid along an alternative pathway towards the waste chamberwhen the fluid reaches the constriction. Small holes in the gasket,together with a hydrophobic coating, are provided to prevent flow fromthe first conduit to the second conduit until sufficient pressure isapplied. Features that control the flow of liquids within and betweenthe conduits of the present invention are herein collectively termedvalves.

One embodiment of the invention, therefore, provides a single-usecartridge with a sample holding chamber connected to a first conduitwhich contains an analyte sensor or array of analyte sensors. A secondconduit, partly containing a fluid, is connected to the first conduitand air segments can be introduced into the fluid in the second conduitin order to segment it. Pump means are provided to displace the samplewithin the first conduit, and this displaces fluid from the secondconduit into the first conduit. Thus, the sensor or sensors can becontacted first by a sample and then by a second fluid.

In another embodiment, the cartridge includes a closeable valve locatedbetween the first conduit and a waste chamber. This embodiment permitsdisplacement of the fluid from the second conduit into the first conduitusing only a single pump means connected to the first conduit. Thisembodiment further permits efficient washing of the conduits of thecartridge of the present invention, which is an important feature of asmall single-use cartridge. In operation, the sample is displaced tocontact the sensors, and is then displaced through the closeable valveinto the waste chamber. Upon wetting, the closeable valve seals theopening to the waste chamber, providing an airtight seal that allowsfluid in the second conduit to be drawn into contact with the sensorsusing only the pump means connected to the first conduit. In thisembodiment, the closeable valve permits the fluid to be displaced inthis manner and prevents air from entering the first conduit from thewaste chamber.

In another embodiment, both a closeable valve and means for introducingsegments into the conduit are provided. This embodiment has manyadvantages, among which is the ability to reciprocate a segmented fluidover the sensor or array of sensors. Thus a first segment or set ofsegments is used to rinse a sensor, and then a fresh segment replaces itfor taking measurements. Only one pump means (that connected to thefirst conduit) is required.

In another embodiment, analyte measurements are performed in a thin-filmof liquid coating an analyte sensor. Such thin-film determinations arepreferably performed amperometrically. This cartridge differs from theforegoing embodiments in having both a closeable valve that is sealedwhen the sample is expelled through the valve, and an air vent withinthe conduits that permits at least one air segment to be subsequentlyintroduced into the measuring fluid, thereby increasing the efficiencywith which the sample is rinsed from the sensor, and further permittingremoval of substantially all the liquid from the sensor prior tomeasurement, and still further permitting segments of fresh liquid to bebrought across the sensor to permit sequential, repetitive measurementsfor improved accuracy and internal checks of reproducibility.

The analysis scheme for the detection of low concentrations ofimmunoactive analyte relies on the formation of an enzyme labeledantibody/analyte/surface-bound antibody “sandwich” complex, as discussedabove. The concentration of analyte in a sample is converted into aproportional surface concentration of an enzyme. The enzyme is capableof amplifying the analyte's chemical signal by converting a substrate toa detectable product. For example, where alkaline phosphatase is theenzyme, a single enzyme molecule can produce about nine thousanddetectable molecules per minute, providing several orders of magnitudeimprovement in the detectability of the analyte compared to schemes inwhich an electroactive species is attached to the antibody in place ofalkaline phosphatase.

In immunosensor embodiments, it is advantageous to contact the sensorfirst with a sample and then with a wash fluid prior to recording aresponse from the sensor. In specific embodiments, the sample is amendedwith an antibody-enzyme conjugate (signal antibody) that binds to theanalyte of interest within the sample before the amended sample contactsthe sensor. Binding reactions in the sample produce ananalyte/antibody-enzyme complex. The sensor comprises an immobilizedantibody to the analyte, attached close to an electrode surface. Uponcontacting the sensor, the analyte/antibody-enzyme complex binds to theimmobilized antibody near the electrode surface. It is advantageous atthis point to remove from the vicinity of the electrode as much of theunbound antibody-enzyme conjugate as possible to minimize backgroundsignal from the sensor. The enzyme of the antibody-enzyme complex isadvantageously capable of converting a substrate, provided in the fluid,to produce an electrochemically active species. This active species isproduced close to the electrode and provides a current from a redoxreaction at the electrode when a suitable potential is applied(amperometric operation). Alternatively, if the electroactive species isan ion, it can be measured potentiometrically. In amperometricmeasurements the potential may either be fixed during the measurement,or varied according to a predetermined waveform. For example, atriangular wave can be used to sweep the potential between limits, as isused in the well-known technique of cyclic voltammetry. Alternatively,digital techniques such as square waves can be used to improvesensitivity in detection of the electroactive species adjacent to theelectrode. From the current or voltage measurement, the amount orpresence of the analyte in the sample is calculated. These and otheranalytical electrochemical methods are well known in the art.

In embodiments in which the cartridge comprises an immunosensor, theimmunosensor is advantageously microfabricated from a base sensor of anunreactive metal such as gold, platinum or iridium, and a porouspermselective layer which is overlaid with a bioactive layer attached toa microparticle, for example latex particles. The microparticles aredispensed onto the porous layer covering the electrode surface, formingan adhered, porous bioactive layer. The bioactive layer has the propertyof binding specifically to the analyte of interest, or of manifesting adetectable change when the analyte is present, and is most preferably animmobilized antibody directed against the analyte.

Referring to the Figures, the cartridge of the present inventioncomprises a cover, FIGS. 1 and 2, a base, FIG. 4, and a thin-filmadhesive gasket, FIG. 3, disposed between the base and the cover.Referring now to FIG. 1, the cover 1 is made of a rigid material,preferably plastic, and capable of repetitive deformation at flexiblehinge regions 5, 9, 10 without cracking. The cover comprises a lid 2,attached to the main body of the cover by a flexible hinge 9. Inoperation, after introduction of a sample into the sample holdingchamber 34, the lid can be secured over the entrance to the sample entryport 4, preventing sample leakage, and the lid is held in place by hook3. The cover further comprises two paddles 6, 7, that are moveablerelative to the body of the cover, and which are attached to it byflexible hinge regions 5, 10. In operation, when operated upon by a pumpmeans, paddle 6 exerts a force upon an air bladder comprised of cavity43, which is covered by thin-film gasket 21, to displace fluids withinconduits of the cartridge. When operated by a second pump means, paddle7 exerts a force upon the gasket 21, which can deform because of slits22 cut therein. The cartridge is adapted for insertion into a readingapparatus, and therefore has a plurality of mechanical and electricalconnections for this purpose. It should also be apparent that manualoperation of the cartridge is possible. Thus, upon insertion of thecartridge into a reading apparatus, the gasket transmits pressure onto afluid-containing foil pack filled with approximately 130 μL ofanalysis/wash solution (“fluid”) located in cavity 42, rupturing thepackage upon spike 38, and expelling fluid into conduit 39, which isconnected via a short transecting conduit in the base to the sensorconduit. The analysis fluid fills the front of the analysis conduitfirst pushing fluid onto a small opening in the tape gasket that acts asa capillary stop. Other motions of the analyzer mechanism applied to thecartridge are used to inject one or more segments into the analysisfluid at controlled positions within the analysis conduit. Thesesegments are used to help wash the sensor surface and the surroundingconduit with a minimum of fluid.

The cover further comprises a hole covered by a thin pliable film 8. Inoperation, pressure exerted upon the film expels one or more airsegments into a conduit 20 through a small hole 28 in the gasket.

Referring to FIG. 2, the lower surface of the base further comprisessecond conduit 11, and first conduit 15. Second conduit 11 includes aconstriction 12, which controls fluid flow by providing resistance tothe flow of a fluid. Optional coatings 13, 14 provide hydrophobicsurfaces, which together with gasket holes 31, 32, control fluid flowbetween second and first conduits 11, 15. A recess 17 in the baseprovides a pathway for air in conduit 34 to pass to conduit 34 throughhole 27 in the gasket.

Referring to FIG. 3, thin-film gasket 21 comprises various holes andslits to facilitate transfer of fluid between conduits within the baseand the cover, and to allow the gasket to deform under pressure wherenecessary. Thus, hole 24 permits fluid to flow from conduit 11 intowaste chamber 44; hole 25 comprises a capillary stop between conduits 34and 15; hole 26 permits air to flow between recess 18 and conduit 40;hole 27 provides for air movement between recess 17 and conduit 34; andhole 28 permits fluid to flow from conduit 19 to waste chamber 44 viaoptional closeable valve 41. Holes 30 and 33 permit the plurality ofelectrodes that are housed within cutaways 35 and 37, respectively, tocontact fluid within conduit 15. In a specific embodiment, cutaway 37houses a ground electrode, and/or a counter-reference electrode, andcutaway 35 houses at least one analyte sensor and, optionally, aconductimetric sensor. In FIG. 3 the tape 21 is slit at 22 to allow thetape enclosed by the three cuts 22 to deform when the instrument appliesa downward force to rupture the calibrant pouch within element 42 on thebarb 38. The tape is also cut at 23 and this allows the tape to flexdownwards into element 43 when the instrument provides a downward force,expelling air from chamber 43 and moving the sample fluid throughconduit 15 towards the sensors. Element 29 in FIG. 3 acts as an openingin the tape connecting a region in the cover FIG. 2 with the base FIG.4.

Referring to FIG. 4, conduit 34 is the sample holding chamber thatconnects the sample entry port 4 to first conduit 11 in the assembledcartridge. Cutaway 35 houses the analyte sensor or sensors, or ananalyte responsive surface, together with an optional conductimetricsensor or sensors. Cutaway 37 houses a ground electrode if needed as areturn current path for an electrochemical sensor, and may also house anoptional conductimetric sensor. Cutaway 36 provides a fluid path betweengasket holes 31 and 32 so that fluid can pass between the first andsecond conduits. Recess 42 houses a fluid-containing package, e.g., arupturable pouch, in the assembled cartridge that is pierced by spike 38because of pressure exerted upon paddle 7 upon insertion into a readingapparatus. Fluid from the pierced package flows into the second conduitat 39. An air bladder is comprised of recess 43 which is sealed on itsupper surface by gasket 21. The air bladder is one embodiment of a pumpmeans, and is actuated by pressure applied to paddle 6 which displacesair in conduit 40 and thereby displaces the sample from sample chamber34 into first conduit 15.

The location at which air enters the sample holding chamber (gasket hole27) from the bladder, and the capillary stop 25, together define apredetermined volume of the sample holding chamber. An amount of thesample corresponding to this volume is displaced into the first conduitwhen paddle 6 is depressed. This arrangement is therefore one possibleembodiment of a metering means for delivering a metered amount of anunmetered sample into the conduits of the cartridge.

In the present cartridge, a means for metering a sample segment isprovide in the base plastic part. The segment size is controlled by thesize of the compartment in the base and the position of the capillarystop and air pipe holes in the tape gasket. This volume can be readilyvaried from 2 to 200 μL. Expansion of this range of sample sizes ispossible within the context of the present invention.

The fluid is pushed through a pre-analytical conduit 11 that can be usedto amend a reagent (e.g., particles, soluble molecules, or the IgM orfragments thereof) into the sample prior to its presentation at thesensor conduit 19. Alternatively, the amending reagent may be located inportion 15, beyond portion 16. Pushing the sample through thepre-analytical conduit also serves to introduce tension into thediaphragm pump paddle 7 which improves its responsiveness for actuationof fluid displacement.

In some assays, metering is advantageous if quantification of theanalyte is required. A waste chamber is provided, 44, for sample and/orfluid that is expelled from the conduit, to prevent contamination of theoutside surfaces of the cartridge. A vent connecting the waste chamberto the external atmosphere is also provided, 45. One desirable featureof the cartridge is that once a sample is loaded, analysis can becompleted and the cartridge discarded without the operator or otherscontacting the sample.

Referring now to FIG. 5, a schematic diagram of the features of acartridge and components is provided, wherein 51-57 are portions of theconduits and sample chamber that can optionally be coated with dryreagents to amend a sample or fluid. The sample or fluid is passed atleast once over the dry reagent to dissolve it. Reagents used to amendthe sample may include one or more of the following: antibody-enzymeconjugates (signal antibodies), IgM and/or fragments thereof, IgG and/orfragments thereof, and other blocking agents that prevent eitherspecific or non-specific binding reactions among assay compounds. Asurface coating that is not soluble but helps prevent non-specificadsorption of assay components to the inner surfaces of the cartridgescan also be provided.

In specific embodiments, a closeable valve is provided between the firstconduit and the waste chamber. In one embodiment, this valve, 58, iscomprised of a dried sponge material that is coated with an impermeablesubstance. In operation, contacting the sponge material with the sampleor a fluid results in swelling of the sponge to fill the cavity 41 (FIG.4), thereby substantially blocking further flow of liquid into the wastechamber 44. Furthermore, the wetted valve also blocks the flow of airbetween the first conduit and the waste chamber, which permits the firstpump means connected to the sample chamber to displace fluid within thesecond conduit, and to displace fluid from the second conduit into thefirst conduit in the following manner.

Referring now to FIG. 6, which illustrates the schematic layout of animmunosensor cartridge, there are provided three pumps, 61-63. Whilethese pumps have been described in terms of specific embodiments, itwill be readily understood that any pumping device capable of performingthe respective functions of pumps 61-63 may be used within the presentinvention. Thus, pump 1, 61, should be capable of displacing the samplefrom the sample holding chamber into the first conduit; pump 2, 62,should be capable of displacing fluid within the second conduit; andpump 3, 63, should be capable of inserting at least one segment into thesecond conduit. Other types of pumps that are envisaged in the presentapplication include, but are not limited to, an air sac contacting apneumatic means whereby pressure is applied to the air sac, a flexiblediaphragm, a piston and cylinder, an electrodynamic pump, and a sonicpump. With reference to pump 3, 63, the term “pump” includes all devicesand methods by which one or more segments are inserted into the secondconduit, such as a pneumatic means for displacing air from an air sac, adry chemical that produces a gas when dissolved, or a plurality ofelectrolysis electrodes operably connected to a current source. In aspecific embodiment, the segment is produced using a mechanical segmentgenerating diaphragm that may have more than one air bladder or chamber.As shown, the well 8 has a single opening which connects the innerdiaphragm pump and the fluid filled conduit into which a segment is tobe injected 20. The diaphragm can be segmented to produce multiplesegments, each injected in a specific location within a fluid filledconduit. In FIG. 6, element 64 indicates the region where theimmunosensor performs the capture reaction to form a sandwich comprisingthe immobilized antibody, the analyte and the signal antibody.

In alternative embodiments, a segment is injected using a passivefeature. A well in the base of the cartridge is sealed by the tapegasket. The tape gasket covering the well has two small holes on eitherend. One hole is open while the other is covered with a filter materialwhich wets upon contact with a fluid. The well is filled with a loosehydrophilic material such as a cellulose fiber filter, paper filter orglass fiber filter. This hydrophilic material draws the liquid into thewell in the base via capillary action, displacing the air that wasformerly in the well. The air is expelled through the opening in thetape gasket creating a segment whose volume is determined by the volumeof the well and the void volume of the loose hydrophilic material. Thefilter used to cover one of the inlets to the well in the base can bechosen to meter the rate at which the fluid fills the well and therebycontrol the rate at which the segment is injected into the conduit inthe cover. This passive feature permits any number of controlledsegments to be injected at specific locations within a fluid path andrequires a minimum of space.

Based on the present disclosure it is apparent the present methodprovides a way of reducing or eliminating interference from heterophileantibodies in an analyte immunoassay where a sample, e.g., whole bloodsample, is first collected and then amended by dissolving a dry reagentcomprising IgM or fragments thereof into the sample. This yields asample with a dissolved non-human IgM concentration of at least about 20μg/mL, which is sufficient to substantially sequester any heterophileantibodies in the sample. Once this step is completed, it is possible toperform an immunoassay, e.g., an electrochemical immunoassay, on theamended sample to determine the concentration of an analyte.

Note that the dissolution of the dry reagent and the sandwich formationstep can occur concurrently or in a stepwise manner. The method isdirected mainly to analytes that are cardiovascular markers, e.g., TnI,TnT, CKMB, myoglobin, BNP, NT-proBNP, and proBNP, but can also be usedfor other markers such as, for example, beta-HCG, TSH, D-dimer, and PSA.To ensure that the majority of the heterophile antibodies aresequestered before the detection step, it is preferable that the sampleamendment step is for a selected predetermined period in the range ofabout 1 minute to about 30 minutes.

The method is preferably performed in a cartridge comprising animmunosensor, a conduit, a sample entry port and a sample holdingchamber, where at least a portion of at least one of these elements iscoated with the dry reagent. Note that the dry reagent can includebuffer, salt, surfactant, stabilizing agent, a simple carbohydrate, acomplex carbohydrate and various combinations. In addition the dryreagent can also include an enzyme-labeled antibody (signal antibody) tothe analyte.

For a TnI and BNP assays, the dry reagent preferably dissolves into thesample to give an IgM concentration (or equivalent IgM fragmentconcentration) of at least 20 μg/mL, e.g., at least 30 μg/mL, at least40 μg/mL, or at least 50 μg/mL. In terms of ranges, the dry reagentpreferably dissolves into the sample to give an IgM concentration (orequivalent IgM fragment concentration) of from about 20 μg/mL to about200 μg/mL, preferably from about 20 μg/mL to about 60 μg/mL.

In preferred embodiments, e.g., TnI and BNP assays, IgG or fragmentsthereof are used in combination with the IgM or the IgM fragments. Thus,for example, the dry reagent coating may further comprise IgG orfragments of IgG in an amount sufficient to dissolve into the sample togive an IgG concentration or equivalent IgG fragment concentrationgreater than about 50 μg/mL, e.g., greater than about 100 μg/mL, greaterthan about 250 μg/mL or greater than about 500 μg/mL. In terms ofranges, the amending with IgG or fragments thereof preferably yields anIgG concentration or equivalent IgG fragment concentration of from about50 μg/mL to about 5000 μg/mL, preferably from about 500 μg/mL to about1000 μg/mL. Thus, in some preferred embodiments, a dry reagent dissolvesinto the sample to give an IgM concentration of from about 20 to about200 μg/mL, preferably from about 20 μg/mL to about 60 μg/mL, and an IgGconcentration of from about 50 to about 5000 μg/mL, preferably fromabout 500 μg/mL to about 1000 μg/mL.

In those embodiments in which the biological sample, e.g., whole blood,is amended with both IgG or fragments thereof and IgM or fragmentsthereof, the IgM or fragments thereof and the IgG or fragments thereofpreferably are added at a weight ratio greater than 0.004, e.g., greaterthan 0.02, greater than 0.05 or greater than 0.1. In terms of ranges,the weight ratio of IgM or fragments thereof to IgG or fragments thereofpreferably is from 0.004 to 4, e.g., from 0.02 to 2, or from 0.05 to0.15. These weight ratios apply to the desired amending medium, e.g.,dry coating layer, as well as in the resulting amended samples.

As suggested above, in addition to or instead of using whole IgMmolecules, which comprise pentamers where the individual monomers areformed from an Fc region attached to a F(ab′)2 region, which in turncomprises two Fab regions, it is also possible to use fragments of IgM.IgM fragmentation can be achieved variously using combinations ofdisulphide bond reduction (—S—S— to —SH HS—) and enzymatic pepsin orpapain digestion, to create some combination of F(ab′)2 fragments, Fabfragments, and/or Fc fragments. These fragments can be separated for useseparately by chromatography, or used in combination. For example, wherethe blocking site is on the Fc fragment, this can be used instead of thewhole IgM molecule. The same applies to the Fab fragment and the F(ab′)2fragment. Where this approach is used it is desirable that the essentialfragment molar concentration is similar to, e.g., the equivalent of,that of the native IgM pentamer. As previously described, the preferredembodiment of the invention uses IgM in a concentration of at leastabout 20 μg/mL. Since the IgM (pentamer) has a molecular weight of about900 KD, this is equivalent to an IgM concentration of about 22.2 nM.Where IgM fragments are used it is desirable to account for thefive-fold molar difference in Fc and F(ab′)2 and ten-fold molardifference in Fab fragments, to attain substantially the same level ofblocking.

In the actual assay step, it is preferred that once the sandwich isformed between the immobilized and signal antibodies, the sample mediumis subsequently washed to a waste chamber, followed by exposing thesandwich to a substrate capable of reacting with an enzyme to form aproduct capable of electrochemical detection. The preferred format is anelectrochemical enzyme-linked immunosorbent assay.

Preferably, the device is one that performs an immunoassay of an analytein a sample, e.g., blood sample, with reduced interference fromheterophile antibodies. The device is a housing with an electrochemicalimmunosensor, a conduit and a sample entry port, where the conduitpermits the sample to pass from the entry port to the immunosensor in acontrolled manner. In one aspect, at least a portion of the housing iscoated with a dry reagent which can comprise a non-human IgM and/orfragments thereof, or a mixture of non-human IgG and non-human IgMand/or fragments thereof. The important feature is that the dry reagentis capable of dissolving into the sample to yield an IgM concentrationor equivalent IgM fragment concentration of at least about 20 μg/mL.This is generally sufficient to sequester any heterophile antibodies inthe sample. In a preferred embodiment, the device also comprises animmuno-reference sensor. The immunosensor is preferably directed todetect a cardiovascular marker, e.g., analytes such as TnI, TnT, CKMB,myoglobin, BNP, NT-proBNP, and proBNP. The system in which the deviceoperates generally allows the sample to remain in contact with thereagent for a predetermined period, e.g., from 1 to 30 minutes.Preferably the device is a single-use cartridge, e.g., filled with asingle sample, used once for the test and then discarded. Generally, thedevice includes a wash fluid capable of washing the sample to a wastechamber, and a substrate capable of reacting with the enzyme sandwich atthe immunosensor to form a product suitable for electrochemicaldetection.

More broadly the invention relates to reducing interference fromheterophile antibodies in an analyte immunoassay for any biologicalsample, e.g. samples including whole blood, serum, plasma, urine anddiluted forms thereof. In addition, the way for amending the sample toyield a given non-human IgM concentration can be based on dissolving adry reagent or by adding a solution containing IgM. Furthermore,performing an immunoassay on the amended sample to determine theconcentration of the selected analyte can be based on various techniquesincluding electrochemical ones, e.g., amperometric and potentiometric,and also optical ones, e.g., absorbance, fluorescence and luminescence.

The present invention will be better understood in view of the followingnon-limiting Examples.

EXAMPLE 1

FIG. 7 illustrates the principle of an amperometric immunoassayaccording to specific embodiments of the present invention fordetermining the presence and amount of troponin I (TnI), a marker ofcardiac function. A blood sample was introduced into the sample holdingchamber of a cartridge of the present invention, and was amended bydissolution of a dry reagent coated into the sample holding chamber. Thedry reagent includes IgM 77, as described above, which upon dissolutioninto the sample selectively binds to complementary heterophileantibodies 78 that may be contained in the sample. In other embodiments,not shown, fragments derived from IgM may be employed to sequesterheterophile antibodies contained in the sample. As shown, the dryreagent also comprises IgG 79, which also selectively binds tocomplementary antibodies 78 after dissolution into the sample. In otherembodiments, fragments derived from IgG may be employed to sequesterheterophile antibodies contained in the sample.

In addition, a conjugate molecule comprising alkaline phosphatase enzyme(AP) covalently attached to a polyclonal anti-troponin I antibody (aTnI)71 (signal antibody) also was dissolved into the sample. This conjugatespecifically binds to the TnI, 70, in the blood sample, producing acomplex made up of TnI bound to the AP-aTnI conjugate. In a capturestep, this complex binds to the capture aTnI antibody 72 (immobilizedantibody) attached on, or close to, the immunosensor. The sensor chiphas a conductivity sensor which is used to monitor when the samplereaches the sensor chip. The time of arrival of the fluid can be used todetect leaks within the cartridge: a delay in arrival signals a leak.The position of the sample segment within the sensor conduit can beactively controlled using the edge of the fluid as a position marker. Asthe sample/air interface crosses the conductivity sensor, a precisesignal is generated which can be used as a fluid marker from whichcontrolled fluid excursions can be executed. The fluid segment ispreferentially oscillated edge-to-edge over the sensor in order topresent the entire sample to the immunosensor surface. A second reagentcan be introduced in the sensor conduit beyond the sensor chip, whichbecomes homogenously distributed during the fluid oscillations.

The sensor chip contains a capture region or regions coated withantibodies for the analyte of interest. These capture regions aredefined by a hydrophobic ring of polyimide or anotherphotolithographically produced layer. A microdroplet or severalmicrodroplets (approximately 5 to 40 nanoliters in size) containingantibodies in some form, for example bound to latex microspheres, isdispensed on the surface of the sensor or on a permselective layer onthe sensor. The photodefined ring contains this aqueous droplet allowingthe antibody coated region to be localized to a precision of a fewmicrons. The capture region can be made from 0.03 to roughly 2 squaremillimeters in size. The upper end of this size is limited by the sizeof the conduit and sensor in present embodiments, and is not alimitation of the invention.

Thus, the gold electrode 74 is coated with a biolayer 73 comprising acovalently attached anti-troponin I antibody, to which the TnI/AP-aTnIcomplex binds. AP is thereby immobilized close to the electrode inproportion to the amount of TnI initially present in the sample. Inaddition to specific binding, the enzyme-antibody conjugate may bindnon-specifically to the sensor. NSB provides a background signal fromthe sensor that is undesirable and preferably is minimized. As describedabove, the rinsing protocols, and in particular the use of segmentedfluid to rinse the sensor, provide efficient means to minimize thisbackground signal. In a second step subsequent to the rinsing step, asubstrate 75 that is hydrolyzed by, for example, alkaline phosphatase toproduce an electroactive product 76 is presented to the sensor. Inspecific embodiments the substrate is comprised of a phosphorylatedferrocene or p-aminophenol. The amperometric electrode is either poisedat a fixed electrochemical potential sufficient to oxidize or reduce aproduct of the hydrolyzed substrate but not the substrate directly, orthe potential is swept one or more times through an appropriate range.Optionally, a second electrode may be coated with a layer where thecomplex of TnI/AP-aTnI is made during manufacture to act as a referencesensor or calibration means for the measurement.

In the present example, the sensor comprises two amperometric electrodeswhich are used to detect the enzymatically produced 4-aminophenol fromthe reaction of 4-aminophenylphosphate with the enzyme label alkalinephosphatase. The electrodes are preferably produced from gold surfacescoated with a photodefined layer of polyimide. Regularly spaced openingin the insulating polyimide layer define a grid of small gold electrodesat which the 4-aminophenol is oxidized in a two electron per moleculereaction. Sensor electrodes further comprise a biolayer, while referenceelectrodes can be constructed, for example, from gold electrodes lackinga biolayer, or from silver electrodes, or other suitable material.Different biolayers can provide each electrode with the ability to sensea different analyte.

Substrates, such as p-aminophenol species, can be chosen such that theE(½) of the substrate and product differ substantially. Preferably, thevoltammetric half-wave potential E(½) of the substrate is substantiallyhigher (more positive) than that of the product. When the condition ismet, the product can be selectively electrochemically measured in thepresence of the substrate.

The size and spacing of the electrode play an important role indetermining the sensitivity and background signal. The importantparameters in the grid are the percentage of exposed metal and thespacing between the active electrodes. The position of the electrode canbe directly underneath the antibody capture region or offset from thecapture region by a controlled distance. The actual amperometric signalof the electrodes depends on the positioning of the sensors relative tothe antibody capture site and the motion of the fluid during theanalysis. A current at the electrode is recorded that depends upon theamount of electroactive product in the vicinity of the sensor.

The detection of alkaline phosphatase activity in this example relies ona measurement of the 4-aminophenol oxidation current. This is achievedat a potential of about +60 mV versus the Ag/AgCl ground chip. The exactform of detection used depends on the sensor configuration. In oneversion of the sensor, the array of gold microelectrodes is locateddirectly beneath the antibody capture region. When the analysis fluid ispulled over this sensor, enzyme located on the capture site converts the4-aminophenylphosphate to 4-aminophenol in an enzyme limited reaction.The concentration of the 4-aminophenylphosphate is selected to be inexcess, e.g., 10 times the Km value. The analysis solution is 0.1 M indiethanolamine, 1.0 M NaCl, buffered to a pH of 9.8. Additionally, theanalysis solution contains 0.5 mM MgCl₂, which is a cofactor for theenzyme. Alternatively, a carbonate buffer has the desired properties.

In another electrode geometry embodiment, the electrode is located a fewhundred microns away from the capture region. When a fresh segment ofanalysis fluid is pulled over the capture region, the enzyme productbuilds with no loss due to electrode reactions. After a time, thesolution is slowly pulled from the capture region over the detectorelectrode resulting in a current spike from which the enzyme activitycan be determined.

An important consideration in the sensitive detection of alkalinephosphatase activity is the non-4-aminophenol current associated withbackground oxidations and reductions occurring at the gold sensor. Goldsensors tend to give significant oxidation currents in basic buffers atthese potentials. The background current is largely dependent on thebuffer concentration, the area of the gold electrode (exposed area),surface pretreatments and the nature of the buffer used. Diethanolamineis a particularly good activating buffer for alkaline phosphatase. Atmolar concentrations, the enzymatic rate is increased by about threetimes over a non-activating buffer such as carbonate.

In alternative embodiments, the enzyme conjugated to an antibody orother analyte-binding molecule is urease, and the substrate is urea.Ammonium ions produced by the hydrolysis of urea are detected in thisembodiment by the use of an ammonium sensitive electrode.Ammonium-specific electrodes are well-known to those of skill in theart. A suitable microfabricated ammonium ion-selective electrode isdisclosed in U.S. Pat. No. 5,200,051, incorporated herein by reference.Other enzymes that react with a substrate to produce an ion are known inthe art, as are other ion sensors for use therewith. For example,phosphate produced from an alkaline phosphatase substrate can bedetected at a phosphate ion-selective electrode.

Referring now to FIG. 8, there is illustrated the construction of anembodiment of a microfabricated immunosensor. Preferably a planarnon-conducting substrate 80 is provided onto which is deposited aconducting layer 81 by conventional means or microfabrication known tothose of skill in the art. The conducting material is preferably a noblemetal such as gold or platinum, although other unreactive metals such asiridium may also be used, as may non-metallic electrodes of graphite,conductive polymer, or other materials. An electrical connection 82 isalso provided. A biolayer 83 is deposited onto at least a portion of theelectrode. In the present disclosure, a biolayer means a porous layercomprising on its surface a sufficient amount of a molecule 84 that caneither bind to an analyte of interest, or respond to the presence ofsuch analyte by producing a change that is capable of measurement.Optionally, a permselective screening layer may be interposed betweenthe electrode and the biolayer to screen electrochemical interferents asdescribed in U.S. Pat. No. 5,200,051.

In specific embodiments, a biolayer is constructed from latex beads ofspecific diameter in the range of about 0.001 to 50 microns. The beadsare modified by covalent attachment of any suitable molecule consistentwith the above definition of a biolayer. Many methods of attachmentexist in the art, including providing amine reactiveN-hydroxysuccinimide ester groups for the facile coupling of lysine orN-terminal amine groups of proteins. In specific embodiments, thebiomolecule is chosen from among ionophores, cofactors, polypeptides,proteins, glycopeptides, enzymes, immunoglobulins, antibodies, antigens,lectins, neurochemical receptors, oligonucleotides, polynucleotides,DNA, RNA, or suitable mixtures. In most specific embodiments, thebiomolecule is an antibody selected to bind one or more of humanchorionic gonadotrophin, troponin I, troponin T, troponin C, a troponincomplex, creatine kinase, creatine kinase subunit M, creatine kinasesubunit B, myoglobin, myosin light chain, or modified fragments ofthese. Such modified fragments are generated by oxidation, reduction,deletion, addition or modification of at least one amino acid, includingchemical modification with a natural moiety or with a synthetic moiety.Preferably, the biomolecule binds to the analyte specifically and has anaffinity constant for binding analyte ligand of about 10⁻⁷ to 10⁻¹⁵ M.

In one embodiment, the biolayer, comprising beads having surfaces thatare covalently modified by a suitable molecule, is affixed to the sensorby the following method. A microdispensing needle is used to depositonto the sensor surface a small droplet, preferably about 20 nL, of asuspension of modified beads. The droplet is permitted to dry, whichresults in a coating of the beads on the surface that resistsdisplacement during use.

In addition to immunosensors in which the biolayer is in a fixedposition relative to an amperometric sensor, the present invention alsoenvisages embodiments in which the biolayer is coated upon particlesthat are mobile. The cartridge can contain mobile microparticles capableof interacting with an analyte, for example magnetic particles that arelocalized to an amperometric electrode subsequent to a capture step,whereby magnetic forces are used to concentrate the particles at theelectrode for measurement. One advantage of mobile microparticles in thepresent invention is that their motion in the sample or fluidaccelerates binding reactions, making the capture step of the assayfaster. For embodiments using non-magnetic mobile microparticles, aporous filter is used to trap the beads at the electrode.

Referring now to FIG. 9, there is illustrated a mask design for severalelectrodes upon a single substrate. By masking and etching techniques,independent electrodes and leads can be deposited. Thus, a plurality ofimmunosensors, 94 and 96, and conductimetric sensors, 90 and 92, areprovided in a compact area at low cost, together with their respectiveconnecting pads, 91, 93, 95, and 97, for effecting electrical connectionto the reading apparatus. In principle, a very large array of sensorscan be assembled in this way, each sensitive to a different analyte oracting as a control sensor or reference immunosensor.

Specifically, immunosensors are prepared as follows. Silicon wafers arethermally oxidized to form approximately a 1 micron insulating oxidelayer. A titanium/tungsten layer is sputtered onto the oxide layer to apreferable thickness of between 100 to 1000 Angstroms, followed by alayer of gold that is most preferably 800 Angstroms thick. Next, aphotoresist is spun onto the wafer and is dried and baked appropriately.The surface is then exposed using a contact mask, such as a maskcorresponding to that illustrated in FIG. 9. The latent image isdeveloped, and the wafer is exposed to a gold-etchant. The patternedgold layer is coated with a photodefinable polyimide, suitably baked,exposed using a contact mask, developed, cleaned in an O₂ plasma, andpreferably imidized at 350° C. for 5 hours. An optional metallization ofthe back side of the wafer may be performed to act as a resistiveheating element, where the immunosensor is to be used in a thermostattedformat. The surface is then printed with antibody-coated particles.Droplets, preferably of about 20 nL volume and containing 1% solidcontent in deionized water, are deposited onto the sensor region and aredried in place by air drying. Optionally, an antibody stabilizationreagent (supplied by SurModica Corp. or AET Ltd) is overcoated onto thesensor.

Drying the particles causes them to adhere to the surface in a mannerthat prevents dissolution in either sample or fluid containing asubstrate. This method provides a reliable and reproducibleimmobilization process suitable for manufacturing sensor chips in highvolume.

EXAMPLE 2

With respect to the method of use of a cartridge, an unmetered fluidsample is introduced into sample holding chamber 34 of a cartridge,through sample entry port 4. Capillary stop 25 prevents passage of thesample into conduit 15 at this stage, and holding chamber 34 is filledwith the sample. Lid 2 or element 200 is closed to prevent leakage ofthe sample from the cartridge. The cartridge is then inserted into areading apparatus, such as that disclosed in U.S. Pat. No. 5,821,399 toZelin, which is hereby incorporated by reference. Insertion of thecartridge into a reading apparatus activates the mechanism whichpunctures a fluid-containing package located at 42 when the package ispressed against spike 38. Fluid is thereby expelled into the secondconduit, arriving in sequence at 39, 20, 12 and 11. The constriction at12 prevents further movement of fluid because residual hydrostaticpressure is dissipated by the flow of fluid via second conduit portion11 into the waste chamber 44. In a second step, operation of a pumpmeans applies pressure to air bladder 43, forcing air through conduit40, through cutaways 17 and 18, and into conduit 34 at a predeterminedlocation 27. Capillary stop 25 and location 27 delimit a metered portionof the original sample. While the sample is within sample holdingchamber 34, it is amended with the dry reagent coating comprising IgM(and/or fragments thereof) and other materials on the inner surface ofthe chamber. The metered portion of the sample is then expelled throughthe capillary stop by air pressure produced within air bladder 43. Thesample passes into conduit 15 and into contact with the analyte sensoror sensors located within cutaway 35.

In embodiments employing an immunosensor located within cutout 35, thesample is amended prior to arriving at the sensor by, for example, anenzyme-antibody conjugate (signal antibody) and the IgM reagent orfragments thereof. To promote efficient binding of the analyte to thesensor, the sample containing the analyte is optionally passedrepeatedly over the sensor in an oscillatory motion. Preferably, anoscillation frequency of between about 0.2 and 2 Hz is used, mostpreferably 0.7 Hz. Thus, the signal enzyme associated with the signalantibody is brought into close proximity to the amperometric electrodesurface in proportion to the amount of analyte present in the sample.

Once an opportunity for the analyte/enzyme-antibody conjugate complex tobind to the immunosensor has been provided, the sample is ejected byfurther pressure applied to air bladder 43, and the sample passes towaste chamber 44. A wash step next removes non-specifically boundenzyme-conjugate from the sensor chamber. Fluid in the second conduct ismoved by a pump means 43, into contact with the sensors. The analysisfluid is pulled slowly until the first air segment is detected at aconductivity sensor.

The air segment or segments can be produced within a conduit by anysuitable means, including but not limited to: (1) passive means, asshown in FIG. 14 and described below; (2) active means including atransient lowering of the pressure within a conduit using a pump wherebyair is drawn into the conduit through a flap or valve; or (3) bydissolving a compound pre-positioned within a conduit that liberates agas upon contacting fluid in the conduit, where such compound mayinclude a carbonate, bicarbonate or the like. This segment is extremelyeffective at clearing the sample-contaminated fluid from conduit 15. Theefficiency of the rinsing of the sensor region is greatly enhanced bythe introduction of one or more air segments into the second conduit asdescribed. The leading and/or trailing edges of air segments are passedone or more times over the sensors to rinse and resuspend extraneousmaterial that may have been deposited from the sample. Extraneousmaterial includes any material other than specifically bound analyte oranalyte/antibody-enzyme conjugate complex. However, it is an object ofthe invention that the rinsing is not sufficiently protracted orvigorous as to promote dissociation of specifically bound analyte oranalyte/antibody-enzyme conjugate complex from the sensor.

A second advantage of introducing air segments into the fluid is tosegment the fluid. For example, after a first segment of the fluid isused to rinse a sensor, a second segment is then placed over the sensorwith minimal mixing of the two segments. This feature further reducesbackground signal from the sensor by more efficiently removing unboundantibody-enzyme conjugate. After the front edge washing, the analysisfluid is pulled slowly until the first air segment is detected at aconductivity sensor. This segment is extremely effective at clearing thesample-contaminated fluid which was mixed in with the first analysisfluid sample. For measurement, a new portion of fluid is placed over thesensors, and the current or potential, as appropriate to the mode ofoperation, is recorded as a function of time.

EXAMPLE 3

Referring now to FIG. 15, there is shown a top view of an immunosensorcartridge. Cartridge 150 comprises a base and a top portion, preferablyconstructed of a plastic. The two portions are connected by a thin,adhesive gasket or thin pliable film. As in previous embodiments, theassembled cartridge comprises a sample holding chamber 151 into which asample containing an analyte of interest is introduced via a sampleinlet 167. A metered portion of the sample is delivered to the sensorchip 153, via the sample conduit 154 (first conduit) as before by thecombined action of a capillary stop 152, preferably formed by a 0.012inch (0.3 mm) laser cut hole in the gasket or film that connects the twoportions of the cartridge, and an entry point 155 located at apredetermined point within the sample holding chamber whereby airintroduced by the action of a pump means, such as a paddle pushing upona sample diaphragm 156. After contacting the sensor to permit binding tooccur, the sample is moved to vent 157, which contains a wickingmaterial that absorbs the sample and thereby seals the vent closed tothe further passage of liquid or air. The wicking material is preferablya cotton fiber material, a cellulose material, or other hydrophilicmaterial having pores. It is important in the present application thatthe material is sufficiently absorbent (i.e., possesses sufficientwicking speed) that the valve closes within a time period that iscommensurate with the subsequent withdrawal of the sample diaphragmactuating means described below, so that sample is not subsequentlydrawn back into the region of the sensor chip.

As in the specific embodiment shown, there is provided a wash conduit(second conduit) 158, connected at one end to a vent 159 and at theother end to the sample conduit at a point 160 of the sample conduitthat is located between vent 157 and sensor chip 153. Upon insertion ofthe cartridge into a reading apparatus, a fluid is introduced intoconduit 158. Preferably, the fluid is present initially within a foilpouch 161 that is punctured by a pin when an actuating means appliespressure upon the pouch. There is also provided a short conduit 162 thatconnects the fluid to conduit 154 via a small opening in the gasket 163.A second capillary stop initially prevents the fluid from reachingcapillary stop 160, so that the fluid is retained within conduit 158.

After vent 157 has closed, the pump is actuated, creating a loweredpressure within conduit 154. Air vent 164, preferably comprising a smallflap cut in the gasket or a membrane that vibrates to provide anintermittent air stream, provides a means for air to enter conduit 158via a second vent 165. The second vent 165 preferably also containswicking material capable of closing the vent if wetted, which permitssubsequent depression of sample diaphragm 156 to close vent 165, ifrequired. Simultaneously with the actuation of sample diaphragm 156,fluid is drawn from conduit 158, through capillary stop 160, intoconduit 154. Because the flow of fluid is interrupted by air enteringvent 164, at least one air segment (a segment or stream of segments) isintroduced.

Further withdrawal of sample diaphragm 156 draws the liquid containingat least one air segment back across the sensing surface of sensor chip153. The presence of air-liquid boundaries within the liquid enhancesthe rinsing of the sensor chip surface to remove remaining sample.Preferably, the movement of the sample diaphragm 156 is controlled inconjunction with signals received from the conductivity electrodeshoused within the sensor chip adjacent to the analyte sensors. In thisway, the presence of liquid over the sensor is detected, and multiplereadings can be performed by movement of the fluid in discrete steps.

It is advantageous in this embodiment to perform analyte measurementswhen only a thin film of fluid coats the sensors, ground chip 165, and acontiguous portion of the wall of conduit 154 between the sensors andground electrode. A suitable film is obtained by withdrawing fluid byoperation of the sample diaphragm 156, until the conductimetric sensorlocated next to the sensor indicates that bulk fluid is no longerpresent in that region of conduit 154. It has been found thatmeasurement can be performed at very low (nA) currents, the potentialdrop that results from increased resistance of a thin film betweenground chip and sensor chip (compared to bulk fluid), is notsignificant.

The ground chip 165 is preferably silver/silver chloride. It isadvantageous, to avoid air segments, which easily form upon therelatively hydrophobic silver chloride surface, to pattern the groundchip as small regions of silver/silver chloride interspersed with morehydrophilic regions, such as a surface of silicon dioxide. Thus, apreferred ground electrode configuration comprises an array ofsilver/silver chloride squares densely arranged and interspersed withsilicon dioxide. There is a further advantage in the avoidance ofunintentional segments if the regions of silver/silver chloride aresomewhat recessed.

Referring now to FIG. 16, there is shown a schematic view of thefluidics of the preferred embodiment of an immunosensor cartridge.Regions R1-R7 represent specific regions of the conduits associated withspecific operational functions. Thus R1 represents the sample holdingchamber; R2 the sample conduit whereby a metered portion of the sampleis transferred to the capture region, and in which the sample isoptionally amended with a substance coated upon the walls of theconduit; R3 represents the capture region, which houses theconductimetric and analyte sensors; R4 and R5 represent portions of thefirst conduit that are optionally used for further amendment of fluidswith substances coated onto the conduit wall, whereby more complex assayschemes are achieved; R6 represents the portion of the second conduitinto which fluid is introduced upon insertion of the cartridge into areading apparatus; R7 comprises a portion of the conduit located betweencapillary stops 160 and 166, in which further amendment can occur; andR8 represents the portion of conduit 154 located between point 160 andvent 157, and which can further be used to amend liquids containedwithin.

EXAMPLE 4

With regard to the coordination of fluidics and analyte measurements,during the analysis sequence, a user places a sample into the cartridge,places the cartridge into the analyzer and in from 1 to 20 minutes, aquantitative measurement of one or more analytes is performed. Herein isa non-limiting example of a sequence of events that occur during theanalysis:

(1) A 25 to 50 μL sample is introduced in the sample inlet 167 and fillsto a capillary stop 151 formed by a 0.012 inch (0.3 mm) laser cut holein the adhesive tape holding the cover and base components together. Oneor more dry reagent coatings comprising IgM and/or fragments thereof forameliorating heterophile interference and preferably a signal antibodyare dissolved into the sample. The user rotates a latex rubber diskmounted on a snap flap to close the sample inlet 167 and places thecartridge into the analyzer.

(2) The analyzer makes contact with the cartridge, and a motor drivenplunger presses onto the foil pouch 161 forcing the wash/analysis fluidout into a central conduit 158.

(3) A separate motor driven plunger contacts the sample diaphragm 156pushing a measured segment of the sample along the sample conduit (fromreagent region R1 to R2). The sample is detected at the sensor chip 153via the conductivity sensors. The sensor chip is located in captureregion R3.

(4) The sample is oscillated by means of the sample diaphragm 156between R2 and R5 in a predetermined and controlled fashion for acontrolled time to promote binding to the sensor.

(5) The sample is pushed towards the waste region of the cartridge (R8)and comes in contact with a passive pump 157 in the form of a celluloseor similar absorbent wick. The action of wetting this wick seals thewick to air flow thus eliminating its ability to vent excess pressuregenerated by the sample diaphragm 156. The active vent becomes the“controlled air vent” of FIG. 16.

(6) Rapid evacuation of the sample conduit (effected by withdrawing themotor driven plunger from the sample diaphragm 156) forces a mixture ofair (from the vent) and wash/analysis fluid from the second conduit tomove into the inlet located between R5 and R4 in FIG. 16. By repeatingthe rapid evacuation of the sample conduit, a series of air separatedfluid segments are generated which are pulled across the sensor chiptowards the sample inlet (from R4 to R3 to R2 and R1). This washes thesensor free of excess reagents and wets the sensor with reagentsappropriate for the analysis. The wash/analysis fluid which originatesin the foil pouch can be further amended by addition of reagents in R7and R6 within the central wash/analysis fluid conduit.

(7) The wash/analysis fluid segment is drawn at a slower speed towardsthe sample inlet to yield a sensor chip which contains only a thin layerof the analysis fluid. The electrochemical analysis is performed at thispoint. The preferred method of analysis is amperometry but potentiometryor impedance detection is also used.

(8) And the mechanism retracts allowing the cartridge to be removed fromthe analyzer.

EXAMPLE 5

In some embodiments, the device employs an immuno-reference sensor forpurposes of assessing the degree of NSB occurring during an assay. Theimmuno-reference sensor is fabricated in much the same way as theanalyte immunosensor with the exception that the immuno reagent is ananti-HSA (human serum albumin) antibody rather than an anti-analyteantibody. Upon exposure to a human whole blood or plasma sample, thereference sensor becomes coated with specifically bound HSA, an abundantendogenous protein present in all human blood samples thus affording acommon reference for all individual tests run using the presentimmunoassay format. NSB arising due to inadequate washing or due to thepresence of interferences can be monitored by means of this secondsensor.

The net signal from the assay is comprised of the specific signalarising from the analyte immunosensor corrected by subtracting thenon-specific signal arising from the reference sensor, e.g., NetSignal=Analyte Sensor Signal−Reference Sensor Signal−Offset, as shown inequation 4 above. The “offset” is a coefficient that accounts for thedifference in the tendency of the two sensors to be subject to NSB. Ineffect, it accounts for the relative “stickiness” of each sensor withrespect to their ability to bind conjugate non-specifically and isestablished based on the responses of samples that are free of analyteand free of interference. This is done by independent experimentation.

The amount of signal tolerated at the reference sensor is subject tolimits defined by a quality control algorithm that seeks to safeguardthe integrity of results at low analyte concentration where the effectsof NSB have the greatest potential to affect assay results in a mannerthat can alter decision-making in a clinical environment. The essentialprincipal is that the existence of excessive signal at the referencesensor acts as a flag for the presence of NSB, due either to aninadequate wash step or interference.

FIG. 17 shows the immunosensor response as a function of the IgMconcentration in the sample inlet (sample holding chamber) printed dryreagent for three normal healthy donors, two with high levels ofheterophile antibody activity (Donors A and B) and one with a lowheterophile antibody activity (Donor C). Specifically, FIG. 17A showsthe response of the cTnI immunosensor signal, FIG. 17B the associatedimmuno-reference sensor, and FIG. 17C the net assay signal (analytesignal−reference signal). All data are collected over the range of zeroto 100 μg/mL of IgM dissolved into the sample. It is clear from thesefigures that an IgM concentration above about 20 μg/mL substantiallyameliorates the effect of heterophile antibodies on these samples.

Those skilled in the art will recognize that the diminishing of signalat both the analyte and reference sensors is evident in these samplesupon the addition of IgM and demonstrates the relative non-specificityassociated with the action of anti-animal/heterophile antibodies onimmunoreagents prepared from antibodies raised in animal species.Furthermore, as the samples employed were obtained in normal, nominallyhealthy individuals outside of a clinical environment, it is a testamentto the relatively ubiquitous nature of these interferences in thegeneral population.

Based on the size of the donor pool (approximately 200 individuals) inwhich the relatively extreme interferences in samples from Donors A andB were observed, one may estimate that something on the order of 1% ofindividuals possess significant heterophile interferences that can bemitigated with murine IgM.

Heterophile interferences in general, i.e., beyond those that requireIgM for mitigation, are estimated variously to occur in as much as 40%of the population. See Clinical and Laboratory Standards Institute(CLSI) Immunoassay Interference by Endogenous Antibodies; ProposedGuideline; CLSI document I/LA30-P (ISBN 1-56238-633-6).

Our studies with samples exhibiting heterophile interference haveindicated that while the majority of these can be neutralized with IgGalone, a smaller subset, perhaps 10-20%, require IgM for mitigation, asdescribed herein. However, given that manufacturers strive to providehigh integrity test results in all cases, it is necessary that thissubset be addressed from the perspective of interferenceneutralization/mitigation. Furthermore, it should be recognized that:(i) study and mitigation of endogenous antibody interference is limitedby the availability of suitable samples, and (ii) there exists thepossibility, if not likelihood, that there are individuals harboringheterophile interferences at levels requiring ever-greaterconcentrations of interference-eliminating reagents in order to produceinterference-free results.

While the impact of heterophile-mitigating reagents is most dramatic inthe case of discrete samples exhibiting interference, the ubiquitousnature of these interferences suggests that improved mitigation may beassociated with a general lowering of variability in an immunoassay whenapplied to a population of individuals. If an array of individualsubjects have variable but discrete levels of heterophile interferencewhich increase variability in test results but which are sufficientlymild as to be undetected by quality control algorithms, diminution ofthe interference would be expected to decrease the overall variabilityassociated with measurement of the population. For example, themeasurement of a reference population of healthy individuals for cardiactroponin levels would be expected to have some dependence on the degreeto which heterophile interferences are neutralized during the course ofthe measurements. A population of 180 nominally healthy individuals,each having undetectable cardiac troponin in circulation, was measuredusing two formulations of interference-eliminating reagent. Oneformulation contained 375 μg/mL IgG while the second formulationcontained 750 μg/mL IgG and 25 μg/mL IgM. The standard deviations of 540measurements of plasma samples from this population were 0.0103 ng/mLand 0.0088 ng/mL for the low and high Ig formulations respectively.

While the present invention as described above is generally directed toreducing or eliminating interference from heterophile antibodies in ananalyte immunoassay with a whole blood sample, it is also applicable toimmunoassays performed in other types of biological samples, e.g.,plasma, serum and urine, and also diluted samples, e.g., blood, plasma,serum and urine diluted with a buffer. Furthermore, while the inventionis generally directed to amending the sample by dissolving into thesample a dry reagent, it is also practical in other embodiments to addthe reagent as a liquid to the sample during the analysis or duringsample collection. It is also apparent that the present invention hasbeen described herein in terms of electrochemical detection approaches,e.g., amperometric and potentiometric approaches, although it is equallyapplicable to other detection modes, notably optical approaches such asluminescence, fluorescence and absorbance based approaches.

While the invention has been described in terms of various preferredembodiments, those skilled in the art will recognize that variousmodifications, substitutions, omissions and changes can be made withoutdeparting from the spirit of the present invention. Accordingly, it isintended that the scope of the present invention be limited solely bythe scope of the following claims.

1. A method of reducing interference from heterophile antibodies in ananalyte immunoassay, comprising: (a) amending a whole blood sample withnon-human IgM or fragments thereof by dissolving into said sample a dryreagent to yield a non-human IgM concentration of at least 20 μg/mL orequivalent fragment concentration; and (b) performing an electrochemicalimmunoassay on the amended sample to determine the concentration of saidanalyte in said sample.
 2. The method of claim 1, further comprising:(c) amending the whole blood sample with IgG or fragments thereof. 3.The method of claim 2, wherein the whole blood sample is amended withIgM.
 4. The method of claim 2, wherein the whole blood sample is amendedwith IgM F(ab′)2 fragments.
 5. The method of claim 2, wherein the wholeblood sample is amended with IgM Fab fragments.
 6. The method of claim2, wherein the whole blood sample is amended with IgM Fc fragments. 7.The method of claim 2, wherein the analyte is a cardiovascular marker.8. The method of claim 2, wherein the immunoassay is for an analyteselected from the group, TnI, TnT, CKMB, myoglobin, BNP, NT-proBNP, andproBNP.
 9. The method of claim 2, wherein the sample is amended for apredetermined period ranging from about 1 minute to about 30 minutes.10. The method of claim 2, wherein the dry reagent further comprises acomponent selected from the group consisting of buffer, salt,surfactant, stabilizing agent, a simple carbohydrate, a complexcarbohydrate and combinations thereof.
 11. The method of claim 2,wherein the non-human IgG and IgM are murine, caprine or a combinationthereof.
 12. The method of claim 2, wherein the electrochemicalimmunoassay is an enzyme-linked sandwich immunoassay.
 13. The method ofclaim 2, wherein the electrochemical immunoassay is performed by animmunosensor.
 14. The method of claim 2, wherein the electrochemicalimmunoassay is performed by an immunosensor and an immuno-referencesensor.
 15. The method of claim 2, wherein the dry reagent furthercomprises an enzyme-labeled antibody to said analyte.
 16. The method ofclaim 2, further comprising: (d) amending the amended sample with anenzyme-labeled antibody to said analyte by dissolving into said amendedsample a second dry reagent comprising said enzyme-labeled antibody,wherein said second dry reagent is separate from the dry reagent thatcontains said IgM or fragments thereof.
 17. The method of claim 2,wherein the electrochemical assay is performed with an immobilizedantibody to the analyte on an electrode.
 18. The method of claim 2,wherein the amended sample further comprises an enzyme-labeled antibodyto said analyte and is contacted with an immobilized antibody to saidanalyte to form a sandwich of said analyte between said immobilized andlabeled antibodies, the method further comprising the steps of washingsaid sample to a waste chamber and exposing said sandwich to a substratecapable of reacting with said enzyme to form a product capable ofelectrochemical detection.
 19. The method of claim 2, wherein the methodis performed at the point of patient care.
 20. The method of claim 2,wherein the electrochemical immunoassay is an enzyme-linkedimmunosorbent assay.
 21. The method of claim 2, wherein the immunoassayis performed in a cartridge comprising an immunosensor, a conduit, asample entry port and a sample holding chamber.
 22. The method of claim21, wherein at least a portion of at least one of said sample entryport, said sample holding chamber, said conduit and said immunosensorare coated with said dry reagent.
 23. The method of claim 2, wherein thedry reagent further comprises said non-human IgG.
 24. The method ofclaim 23, wherein the analyte is TnI, and wherein said dry reagentdissolves into the sample to give an IgM concentration of from about 20to about 200 μg/mL and an IgG concentration of from about 50 to about5000 μg/mL.
 25. The method of claim 23, wherein the analyte is TnI, andwherein said dry reagent dissolves into the sample to give an IgMconcentration of from about 20 to about 60 μg/mL and an IgGconcentration of from about 500 to about 1000 μg/mL.
 26. The method ofclaim 23, wherein the analyte is BNP, and wherein said dry reagentdissolves into the sample to give an IgM concentration of from about 20to about 200 μg/mL and an IgG concentration of from about 50 to about5000 μg/mL.
 27. The method of claim 23, wherein the analyte is BNP, andwherein said dry reagent dissolves into the sample to give an IgMconcentration of from about 20 to about 60 μg/mL and an IgGconcentration of from about 500 to about 1000 μg/mL.
 28. A method ofreducing interference from heterophile antibodies in a cardiac troponinI immunoassay, comprising: (a) amending a whole blood sample with amixture comprising: (i) non-human IgG or IgG fragments, and (ii)non-human IgM or IgM fragments, sufficient to substantially sequesterany heterophile antibodies in said sample, wherein the non-human IgMconcentration in the amended sample is at least about 20 μg/mL orequivalent IgM fragment concentration; and (b) performing anelectrochemical immunoassay on the amended sample.
 29. A method ofreducing interference from heterophile antibodies in a brain natriureticpeptide immunoassay, comprising: (a) amending a sample with a mixturecomprising: (i) non-human IgG or IgG fragments, and (ii) non-human IgMor IgM fragments, sufficient to substantially sequester any heterophileantibodies in said sample, wherein the non-human IgM concentration inthe amended sample is at least about 20 μg/mL or equivalent IgM fragmentconcentration; and (b) performing an electrochemical immunoassay on theamended sample.
 30. A device for performing an immunoassay of an analytein a blood sample with reduced interference from heterophile antibodies,comprising a housing, an electrochemical immunosensor, a conduit and asample entry port, wherein said conduit permits a blood sample to passfrom the entry port to said immunosensor, and wherein at least one ofsaid housing, said entry port, and said conduit includes a dry reagentcomprising non-human IgM or fragments thereof and optionally IgG orfragments thereof, said dry reagent being capable of dissolving intosaid blood sample to yield an IgM concentration of at least about 20μg/mL or equivalent fragment concentration and substantiallysequestering any heterophile antibodies in said sample.
 31. The deviceof claim 30, wherein the dry reagent comprises non-human IgM orfragments thereof and IgG or fragments thereof.
 32. The device of claim31, further comprising a metering system for metering an initial bloodsample to form a metered blood sample.
 33. The device of claim 31,further comprising an immuno-reference sensor.
 34. The device of claim31, wherein the analyte is a cardiovascular marker.
 35. The device ofclaim 31, wherein the immunoassay is for an analyte selected from thegroup, TnI, TnT, CKMB, myoglobin, BNP, NT-proBNP, proBNP, beta-HCG, TSH,D-dimer, and PSA.
 36. The device of claim 31, wherein the sample isamended for a predetermined period in the range from of about 1 minuteto about 30 minutes.
 37. The device of claim 31, wherein the device is asingle-use cartridge.
 38. The device of claim 31, wherein the dryreagent further comprises an enzyme-labeled antibody to the analyte. 39.The device of claim 31, further comprising a second dry reagentcomprising an enzyme-labeled antibody to the analyte, wherein the seconddry reagent is separate from the dry reagent that comprises the IgM orfragments thereof.
 40. The device of claim 31, wherein the dry reagentfurther comprises a component selected from the group consisting ofbuffer, salt, surfactant, stabilizing agent, a simple carbohydrate, acomplex carbohydrate and combinations thereof.
 41. The device of claim31, wherein the non-human IgG or fragments thereof and IgM or fragmentsthereof are murine, caprine or a combination thereof.
 42. The device ofclaim 31, wherein the immunosensor performs an electrochemicalenzyme-linked sandwich immunoassay.
 43. The device of claim 31, whereinthe immunosensor comprises an immobilized antibody to the analyte on anelectrode.
 44. The device of claim 31, wherein the analyte is TnI, andwherein said dry reagent dissolves into the sample to give an IgMconcentration of from about 20 to about 200 μg/mL and an IgGconcentration of from about 50 to about 5000 μg/mL.
 45. The device ofclaim 31, wherein the analyte is TnI, and wherein said dry reagentdissolves into the sample to give an IgM concentration of from about 20to about 60 μg/mL and an IgG concentration of from about 500 to about1000 μg/mL.
 46. The device of claim 31, wherein the analyte is BNP, andwherein said dry reagent dissolves into the sample to give an IgMconcentration of from about 20 to about 200 μg/mL and an IgGconcentration of from about 50 to about 5000 μg/mL.
 47. The device ofclaim 31, wherein the analyte is BNP, and wherein said dry reagentdissolves into the sample to give an IgM concentration of from about 20to about 60 μg/mL and an IgG concentration of from about 500 to about1000 μg/mL.
 48. The device of claim 31, further comprising a wash fluidcapable of washing said sample to a waste chamber.
 49. The device ofclaim 31, further comprising a wash fluid comprising a substrate capableof reacting at said immunosensor to form a product capable ofelectrochemical detection.
 50. A method of reducing interference fromheterophile antibodies in an analyte immunoassay comprising: (a)amending a biological sample with IgM or fragments thereof andoptionally IgG or fragments thereof to yield a non-human IgMconcentration of at least about 20 μg/mL or equivalent fragmentconcentration; and (b) performing an immunoassay on the amended sampleto determine the concentration of said analyte in said sample.
 51. Themethod of claim 50, further comprising: (c) amending the biologicalsample with IgG or fragments thereof.
 52. The method of claim 51,wherein the biological sample is selected from the group consisting ofwhole blood, serum, plasma, urine and diluted forms thereof.
 53. Themethod of claim 51, wherein the biological sample is amended bydissolving into said sample a dry reagent comprising IgM.
 54. The methodof claim 51, wherein the biological sample is amended by dissolving intosaid sample a dry reagent comprising IgM fragments.
 55. The method ofclaim 51, wherein the biological sample is amended by adding a solutioncomprising IgM or fragments thereof.
 56. The method of claim 51, whereinthe immunoassay method is selected from the group consisting ofelectrochemical, amperometric, potentiometric, absorbance, fluorescenceand luminescence.
 57. A method of reducing heterophile antibodyinterference in an analyte immunoassay device, comprising: (a) addingIgM or fragments thereof and IgG or fragments thereof to a biologicalsample in an amount sufficient to substantially sequester anyheterophile antibodies in said sample and forming an amended sample,wherein the IgM or fragments thereof and the IgG or fragments thereofare added at a weight ratio greater than 0.004; and (b) performing anelectrochemical immunoassay on said amended sample to determine theconcentration of the analyte in said amended sample.
 58. The method ofclaim 57, wherein said weight ratio is greater than 0.02.
 59. The methodof claim 57, wherein said weight ratio is greater than 0.05.
 60. Themethod of claim 57, wherein step (a) comprises adding IgM to saidbiological sample.
 61. The method of claim 60, wherein after the addingstep, the IgM is present in said sample in a concentration of at least20 μg/mL.
 62. The method of claim 57, wherein step (a) comprises addingIgM fragments to said biological sample.
 63. The method of claim 62,wherein after the adding step, the IgM fragments are present in saidsample in a concentration equivalent to an IgM concentration of at leastabout 20 μg/mL.
 64. The method of claim 57, wherein the adding comprisesdissolving the IgM or the fragments thereof into said sample from a dryreagent coating contained in the immunoassay device.
 65. The method ofclaim 57, wherein the adding comprises dissolving the IgM or thefragments thereof into said sample from a dry reagent coating containedon a sample collection device.
 66. The method of claim 57, wherein theadding comprises mixing said sample with a liquid comprising said IgM orsaid fragments thereof to form an amended mixture, the method furthercomprising the step of introducing the amended mixture into theimmunoassay device.
 67. A device for performing an immunoassay of ananalyte in a blood sample with reduced interference from heterophileantibodies, comprising a housing, an electrochemical immunosensor, aconduit and a sample entry port, wherein said conduit permits a bloodsample to pass from the entry port to said immunosensor, and wherein atleast one of said housing, said entry port, and said conduit includes adry reagent comprising non-human IgM or fragments thereof and IgG orfragments thereof at a weight ratio greater than 0.004.
 68. The deviceof claim 67, wherein the weight ratio is greater than 0.02.
 69. Thedevice of claim 67, wherein the weight ratio is greater than 0.05.